Studies on the control of mitotic activity

Summary A tetraploid cell population was produced in the primary root meristem of Pisum sativum by one-half hour treatments with various concentrations of colchicine. The tetraploid population so produced was found to be reasonably synchronous in its passage through successive mitotic cycles with the degree of synchrony being more or less proportional to the concentration of colchicine used. The average time between mitoses appears to be of the order of 12 hours which agrees well with previous estimates. Treatments of roots containing tetraploid populations with 2.52 × 10^–5 M. actidione for 15 minutes were used to demonstrate the possibility of using the system for studies on the differential susceptibility of cells at different stages of the mitotic cycle.This work was carried out under contract number RG-4835 of the National Institutes of Health, United States Public Health Service, and an Institutional Grant from the American Cancer Society.Contribution number 59-26 of the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan.Predoctoral Fellow (CF-9871) of the National Cancer Institute, United States Public Health Service     

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia

Summary The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range –0.44––0.73. The Bohr effect is more pronounced at 20°C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

Life history patterns of parental and hybrid Daphnia differ between lakes

1. We investigated whether Daphnia galeata × hyalina hybrids of Lake Constance and Lake Greifensee show the same pattern of life history parameters as previously reported for D. galeata × cucullata hybrids and whether such a pattern is consistent between Daphnia populations from those two lakes. 2. Hybrids in Lake Constance were intermediate in size compared with the parental species. Hybrids in Lake Greifensee were smaller than D. galeata. The intrinsic growth rate (r) of hybrids from Lake Constance was not significantly different from the faster growing parental taxon D. galeata. However, r of hybrids from Lake Greifensee was significantly lower than that of D. galeata. 3. The observed juvenile body length differences between the taxa varied with the clutch number. The first clutch juvenile lengths of the three taxa did not differ for Lake Constance. First clutch juveniles of Lake Greifensee D. galeata were smaller than hybrid first clutch juveniles. The third clutch juvenile length did not differ between taxa from Lake Greifensee, but D. galeata juveniles from Lake Constance were bigger than those of D. hyalina. 4. The life history pattern found in Lake Constance corresponds to previous findings from other studies. The hybrids in this lake combine the faster population growth of one parental species with a relatively small size. In the case of Lake Greifensee hybrids, the relatively large size of first clutch juveniles and the small size of the adults could be interpreted as dual adaptations to invertebrate and fish predation. We speculate that the lower population growth rate of the hybrids is a trade‐off for this twofold protection.     

EPR and multi-field magnetisation of reduced forms of the binuclear iron centre in ribonucleotide reductase from mouse

Mouse ribonucleotide reductase is composed of a 1?:?1 complex of two homodimeric subunits and catalyses the first unique step on the biochemical pathway to DNA synthesis. The small subunit, protein R2, contains dinuclear iron-oxygen clusters and a tyrosyl free radical required for catalytic activity. We have studied the mixed valent and fully reduced forms of the diiron oxygen cluster from mouse R2 protein by low-temperature EPR. EPR signals of the mixed-valent states of proteins R2 reconstituted with ferrous iron and oxygen in normal and deuterated water, using the same buffers, show apparent g values of 1.92, 1.73, and 1.60 for the mixed-valent state in H₂O and 1.93, 1.73, and 1.62 in D₂O. These g values are typical for diiron-oxygen proteins, while the effect of D₂O is unprecedented for this class of proteins. We estimate the coupling constant J for the Heisenberg exchange (H?=?2J*S₁*S₂) to be J?=?–7.5±1?cm^–1 for the mixed-valent form. The diferrous R2 protein shows an integer spin EPR signal in the presence of azide or 20% glycerol. Variable temperature variable field saturation magnetisation measurements show that only in the azide-complexed R2 protein does a weak ferromagnetic coupling occur (J?=?0.26±0.05?cm^–1), while R2 protein in the absence or presence of 20% glycerol contains non-coupled mononuclear ferrous iron (S?=?2) sites.     

A second L-type isozyme of potato glucan phosphorylase: cloning,antisense inhibition and expression analysis

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81–84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1–81) and the insertion sequence (amino acids 463–570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively.Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.     

The response of tomato roots (Lycopersicon esculentum Mill.) to iron deficiency stress: alterations in the pattern of protein synthesis

Dicotyledon plants adapt to iron (Fe) deficiency through a seriesof reactions that increase the ability of the plant to assimilateFe and to increase the efficiency of Fe utilization. In an attemptto gain an insight into these adaptive processes, the specificchanges in protein synthesis associated with the onset of theFe deficiency response in tomato roots (Lycopersicon esculentumMill cv. Rutgers) have been investigated. Roots were grown underFe—sufficient and —deficient conditions, and thepattern of protein synthesis was analysed by in vitro translationof root mRNA and by in vivo labelling of root proteins. Polypeptideswere resolved by two—dimensional polyacrylamide gel elec—trophoresis.Seven polypeptides were identified by in vitro translation,whose synthesis was significantly increased during Fe deficiency.The increase was probably specific to Fe deficiency in thatthe polypep—tide synthesis was not increased during phosphatedeficiency stress, was less prominent following prolonged Fedeficiency and was decreased following re—supply of Feto the hydroponic medium. The pattern of in vitro translation of mRNA isolated from Fe—deficientroots was compared to the results obtainedin vivo followingradiolabelling of proteins. In these analyses, eight polypeptideswere identified, tentatively including the seven polypeptidespreviously identified by in vitro translation. All polypeptideswere characterized with regard to molecular mass and pl andtheir localization in the cell, whether being membrane boundor soluble. It is suggested that members of this group of polypeptidesare involved in the response of the root to Fe deficiency: althoughtheir functions remain to be identified. Key words: In vitro protein synthesis, iron, iron deficiency, root, 2-dimensional PAGE     

Ribonuclease P from plant nuclei and photosynthetic organelles

RNase P consists of both protein and RNA subunits in all organisms and organelles investigated so far, with the exception of chloroplasts and plant nuclei where no enzyme-associated RNA has been detected to date. Studies on substrate specificity revealed that cleavage by plant nuclear RNase P is critically dependent on a complete and intact structure of the substrate. No clearcut answer is yet possible regarding the order of processing events at the 5 or 3 end of tRNAs in the case of nuclear or chloroplast processing enzymes. RNase P from a phylogenetically ancient photosynthetic organelle will be discussed in greater detail: The enzyme from theCyanophora paradoxa cyanelle is the first RNase P from a photosynthetic organelle which has been shown to contain an essential RNA subunit. This RNA is strikingly similar to its counterpart from cyanobacteria, yet it lacks catalytic activity. Properties of the holoenzyme suggest an intermediate position in RNA enzyme evolution, with an eukaryotic-type, inactive RNA and a prokaryotic-type small protein subunit. The possible presence of an RNA component in RNase P from plant nuclei and modern chloroplasts will be discussed, including a critical evaluation of some criteria that have been frequently applied to elucidate the subunit composition of RNase P from different organisms.Abbreviations RNase P Ribonuclease P - (pre-)tRNA transfer ribonucleic acid (precursor) - tRNA ^Ser (- ^Tyr , - ^Phe ) transfer ribonucleic acid specific for serine (tyrosine, phenylalanine) - CyRP RNA RNA component of cyanelle RNase P     

Mapping of the spectral density function of a Cα−Hα bond vector from NMR relaxation rates of a 13C-labelled α-carbon in motilin

Summary The peptide hormone motilin was synthesised with a ¹³C-enriched -carbon in the leucine at position 10. In aqueous solution, six different relaxation rates were measured for the ¹³C–H fragment as a function of temperature and with and without the addition of 30% (v/v) of the cosolvent d ₂-1,1,1,3,3,3-hexafluoro-2-propanol (HFP). The relaxation rates were analysed employing the spectral density mapping technique introduced by Peng and Wagner [(1992) J. Magn. Reson., 98, 308–322] and using the model-free approach by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570]. The fit to various models of dynamics was also considered. Different procedures to evaluate the overall rotational correlation time were compared. A single exponential time correlation function was found to give a good fit to the measured spectral densities only for motilin in 30% (v/v) HFP at low temperatures, whereas at high temperatures in this solvent, and in D₂O at all temperatures, none of the considered models gave an acceptable fit. A new empirical spectral density function was tested and found to accurately fit the experimental spectral density mapping points. The application of spectral density mapping based on NMR relaxation data for specific ¹³C–¹H vector is shown to be a highly useful method to study biomolecular dynamics. Advantages are high sensitivity, high precision and uniform sampling of the spectral density function over the frequency range.Abbreviations CD circular dichroism - NOE nuclear Overhauser enhancement - NOESY two-dimensional NOE spectroscopy - INEPT insensitive nuclei enhanced by polarisation transfer - DANTE delays alternating with nutations for tailored excitation - WALTZ-16 wideband, alternating phase, low-power technique for zero residual splitting - FID Free induction decay - ppm parts per million - TSPA 3-trimethylsilyl-(3,3,2,2-d)-propionic acid - HFP d ₂-1,1,1,3,3,3-hexafluoro-2-propanol - CPMG Carr-Purcell-Meiboom-Gill - TFD time-resolved fluorescence depolarisation - CSA chemical shift anisotropy Partly presented at the symposium Dynamics and Function of Biomolecules, Szeged, Hungary, July 31–August 2, 1993.