Replicative activity of isolated chromatin from proliferating and quiescent early passage and aging cultured mouse cells

Replicative activity of isolated chromatin from late passage cultured mouse cells has been compared to the activities of chromatin preparaions from dividing and quiescent early passage cells. Rates of endogenous DNA synthesis are similar for chromatin from growing or resting cells but this activity is stimulated 2.5-fold in senescent cell chromatin. Chromatin from growing young cells copies exogenously added single stranded DNA at the highest efficiency. Chromatin of senescent cells copies this template at a lower rate and resting young cell chromatin replicates single stranded DNA at the lowest efficiency. Similar relative rates are obtained when activated DNA is copied by the various chromatin preparations. Total activity of DNA polymerase extracted by salt from chromatin is similar for dividing and quiescent young cells but the proportion of DNA polymerase beta is higher in the latter. Elevated activities of DNA polymerases are extracted from chromatin of old cells. It is concluded, therefore, that chromatin-directed replication is differently arrested in non-dividing senescent cells and in quiescent early passage cells. The possible regulatory mechanisms of DNA replication in quiescence and aging are discussed.     

Detection and resolution of closely related satellite DNA sequences by molecular cloning.

Highly repeated satellite DNAs often consist of mixtures of DNAs with closely related repeating sequences. By cloning individual molecules we have resolved the 1.705 g/cm³ satellite DNA of Drosophila melanogaster into two distinct components: poly$\text{d}\text{A-A-G-A-G}\text{T-T-C-T-C}$ and poly$\text{d}\text{A-A-G-A-G-A-G}\text{T-T-C-T-C-T-C}$. The presence of two distinct sequences within this physically homogeneous satellite DNA had not previously been detected by standard physical, chemical, or sequencing techniques. Both cloning and direct sequence analysis suggest that the five-base-pair and seven-base-pair repeating units reside on separate molecules and are not interspersed with each other.     

Alterations of the carrier-mediated transport of an anionic solute,methotrexate, by charged liposomes in Ehrlich ascites tumor cells

Summary Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5mm phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase in influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor,p-chloromercuriphenylsulfonic acid and the Q₁₀ of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in theV _ma ⁱⁿ , while theK _t ⁱⁿ remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.     

Similarity between 5'- and 3'-terminal nucleotide sequences and double-stranded RNA-derived sequences of eukaryotic mRNA

It has been reported recently that parts of the nucleotide sequences present in the 5′- and 3′-terminal regions of cytoplasmic mRNA are derived from double-stranded hairpin structures of heterogeneous nuclear RNA—a putative mRNA precursor (Naora, 1979). In order to explore the nature of double-stranded hairpin structures, using the sequencing data of human and rabbit globin mRNA and hen ovalbumin mRNA, we examined the following possibility: that certain regions of both the 5′- and 3′-terminal nucleotide sequences of mature mRNA were present in double-stranded hairpin structures covalently linked to both sides of the message sequence in the precursor mRNA molecule and that these double-stranded hairpin structures are similar to each other. The results support the above possibility by showing substantial similarity of nucleotide sequences between the 5′- and 3′-terminal regions of these mRNAs in terms of the formation of similar double-stranded hairpin structures.     

Myo-inositol dehydrogenase(s) from Acetomonas oxydans

Summary Experimental studies have been undertaken with a view to isolation of the enzyme(s) responsible for the stereospecific oxidation of myo-inositol. A partial fractionation has been achieved and the properties of this extract examined. Results show that the active enzyme may well have a cytochrome component and there is indication that the stereospecificity ofAcetomonas oxydans results from permease as opposed to dehydrogenase activity. Kinetic experiments suggest that only one type of active enzyme site is involved in the dehydrogenation of myo-inositol.Departments of Chemistry and Applied Biology     

Nucleotide sequences of HS-α satellite DNA from kangaroo rat dipodomys ordii and characterization of similar sequences in other rodents

The most common repeated nucleotide sequences of the highly repetitive satellite HS-α fraction from kangaroo rat Dipodomys ordii was determined using ribosubstitution methods. This sequence was α nucleotides long and represented about 25% of the total HS-α satellite DNA, while the remaining DNA was composed of sequence variants related to the most common sequence. The sequences of the commonest of these variants are reported. Furthermore, the most common repeated sequence was identical to that reported for the α satellite of guinea pig Cavia porcellus. The α satellites of guinea pig, Cavia porcellus, pocket gopher, Thomomys bottae and antelope ground squirrel, Ammospermophilus leucurus, are shown to have sequences in common with the kangaroo rat. This implies that the simplest repeated sequences of mammalian satellite DNAs may persist over much longer evolutionary times than previously thought.Attempts to explain the very rapid quantitative changes in satellites whose sequence is strongly conserved have led us to consider that they might have a role in sympatric speciation. Among the novel features of the model presented is that fluctuations in satellites could be due to “speciation genes.” Such genes would confer a strong selective advantage in certain situations, and could explain the many puzzling instances in which large numbers of new related species have appeared over a short evolutionary span.     

Effect of drying period and soil moisture on egg hatch of the tadpole shrimp (Notostraca: Triopsidae).

Tadpole shrimp (Triops spp.) are potential biological control agents against larval mosquitoes in temporary ponds and flood-irrigated fields. In some rice field situations, however, they may become pests that uproot and eat young rice plants. In cursory observations, it has been reported that tadpole shrimp eggs do not readily hatch on flooding when the soil or substrate containing eggs is moist before flooding. The relationship between drying (moisture content) of soil and tadpole shrimp hatch was determined in studies conducted in mesocosms at the University of California Aquatic and Vector Control Research facilities at Riverside and at Oasis in the Coachella Valley of southern California. In laboratory hatching trials, an increase in hatch of Triops longicaudatus (LeConte) with declining soil moisture content was demonstrated (t = 8.4, P less than 0.001; r2 = 0.76). In field trials in mesocosms at Riverside, egg hatch increased with increased drying period and declining soil moisture content (G = 29.8, P less than 0.01). No hatch of eggs occurred after 3 d of drying, when soil moisture content was high, but a high level of hatching occurred after 7 and 14 d of drying, when soil moisture declined to low levels. At Oasis, soil moisture did not decrease with drying time because of porous soil and capillary action of water from adjacent flooded mesocosms and thick vegetation covering the pond bottoms. Therefore, hatch rates at Oasis were not associated with the length of the drying period (G = 35, P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of leaf nitrite reductase with Methyl Viologen and ferredoxin under controlled redox conditions.

The dexamethasone binding capacity of embryonal carcinoma cells and their differentiated derivatives was investigated. Manipulation of the embryonal carcinoma cell-culture conditions resulted in an unstable reversible expression of the glucocorticoid receptors. Stable expression of the receptors is observed when these cells are induced to differentiate. Cells grown under identical conditions were assayed for their ability to bind epidermal growth factor.     

Isodityrosine, a new cross-linking amino acid from plant cell-wall glycoprotein.

1. Cell-wall hydrolysates from calli of all higher plants tested contained a new phenolic amino acid for which the trivial name isodityrosine is proposed. Isodityrosine was shown to be an oxidatively coupled dimer of tyrosine with the two tyrosine units linked by a diphenyl ether bridge. 2. The amount of isodityrosine in sodium dodecyl sulphate-insoluble cell-wall preparations was proportional to the amount of hydroxyproline. 3. Acidified chlorite split the diphenyl ether bridge of isodityrosine, and concomitantly solubilized the cell-wall glycoprotein. 4. Dithiothreitol inhibited isodityrosine synthesis in vivo, and suppressed in parallel the covalent binding of newly synthesized protein in the cell wall. 5. It is suggested that isodityrosine is an inter-polypeptide cross-link responsible for the insolubility of plant cell-wall glycoprotein.     

EPR spectra of photosystem I and other iron protein components in intact cells of cyanobacteria.

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe3+ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor 'X' of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 +/- 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe3+, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.