Mutagenicity of ptaquiloside, the carcinogen in bracken, and its related illudane-type sesquiterpenes II. Chromosomal aberration tests with cultured mammalian cells

The chromosomal aberration test using a Chinese hamster lung cell line (CHL) was carried out on ptaquiloside and its related compounds, hypoloside B, hypoloside C, illudin M and illudin S. Ptaquiloside induced chromosomal aberrations at doses as low as 4.5 μg/ml (0.0113 mM). The clastogenic effect was ph-dependent. The same activity was observed at a 90-fold higher dose at pH 5.3 in the culture medium compared with the activity at pH 74. or pH 8.0. Both hypoloside B and hypoloside C were also clastogenic at almost the same dose levels as that of ptaquiloside. Illudin M and illudin S were also potet clastogens and induced aberrations at much lower doses than ptaquiloside. These results suggest that the clastogenic effect is involved in the mechanism of carcinogenic potency of ptaquiloside in animals.     

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

Summary HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved relic DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a HRS60-family. Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.     

Sequence similarities between chicken intestinal 110-kDa ATPase and myosin I-like enzymes

We report the partial amino acid sequence of chicken intestinal microvillar 110-kDa protein that, as a complex with calmodulin, has previously been shown to exhibit myosin-like ATPase and actin-binding activities. The sequence shows a high degree of similarity to the sequence of a novel vertebrate myosin I-like heavy chain encoded by a cDNA isolated from bovine intestine. This confirms that the bovine and chicken proteins are the first examples of Acanthamoeba myosin I-like proteins from higher eukaryotes. Comparison of available structural and functional data leads us to postulate that the myosin I family of proteins result from the fusion of a conserved myosin headlike motor domain, with variable COOH-terminal domains responsible for binding to specific intracellular structures.     

Reevaluation of the amino acid sequence of porcine follitropin

We have reevaluated the sequence of porcine follicle-stimulating hormone (pFSH) with more recent protein-sequencing methodology. This has led to revision of the earlier proposed sequence. As with almost all reported gonadotropin -subunits, NH₂-terminal heterogeneity was found in the porcine FSH -subunit (FSH), starting with residue Phe (1), Asp (3), Gly (4), or Thr (7). In the -subunit, there were found to be at least two molecular species, starting with residue Asn (1) (minor 20%) or Cys (3) (major 80%) as NH₂-terminal and ending at residue Glu (108) as COOH-terminal. The net effect of the present revisions is to increase the homology of pFSH with other reported follitropin sequences. Apparent differences in the half-cystine placements in a previous proposal for pFSH compared with other species of FSH are no longer tenable. The half-cystine placements thus remain a constant structural feature throughout the gonadotropin hormones (choriogonadotropin, follitropin, and lutropin).     

Amino acid sequence and function of rebredoxin from Desulfovibrio vulgaris Miyazaki

Rubredoxin was purified from Desulfovibrio vulgaris Miyazaki. It was sequenced and some of its properties determined. Rubredoxin is composed of 52 amino acids. It is highly homologous to that from D. vulgaris Hildenborough. Its N-methionyl residue is partially formalated. The millimolar absorption coefficients of the rubredoxin at 489 nm and 280 are 8.1 and 18.5, respectively, and the standard redox potential is +5 mB, which is slightly higher than those of other rubredoxins. Rubredoxin, as well as cytochrome c-553, was reduced with lactate by the action of lactate dehydrogenase of this organism, and the rection was stimulated with 2-methyl-1, 4-naphthoquinone. It is suggested that rubredoxin, in collaboration with membraous quinone, functions as natural electron carrier for cytoplasmic lactate dehydrogenase of this organism, whereas cytochrome c-553 plays the same role for periplasmic lactate dehydrogenase.     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.     

Effect of abscisic acid on the photosynthetic oxygen evolution in barley chloroplasts

In vivo effect of abscisic acid (ABA) on photosynthetic oxygen evolution was investigated in barley chloroplasts. The most important kinetic parameters of O₂-producing reactions were changed. The results show inhibition of the O₂-flash yields at ABA concentrations of 10 mol/l and 100 mol/l and an increase in the degree of damping of the oscillations. ABA has a marked effect on the distribution of the oxygenevolving centers in S₀ and S₁ states and on sum of the centers (S₀+S₁) estimated according to the Kok model. In addition, the amplitude and the shape of the initial oxygen burst under continuous illumination are also significantly altered. At a concentration of 100 mol/l, ABA strongly inhibits Hill reaction activity measured by DCPIP reduction. The results cannot be explained by the hypothesis of socalled stomata effect. On the other hand, no effects were observed on the investigated parameters in experiments involving ABA applied in vitro to isolated chloroplasts. It is hypothesized that ABA disrupts the granal chloroplasts structure and raises the degree of participation of the cooperative mechanism of O₂-evolution connected with the functioning of PS II centers in the stroma situated thylakoids.Abbreviations DCPIP 2,6-Dichlorophenolindophenol - DCMU 3-(3,4-dichlorophenil)-1,1-dimethylurea - HEPES N-2-Hydroxyethylpiperazine-N-2-ethane sulfonic acid - PSII photosystem II - RubisCO Ribulose-1,5-bis-phosphate carboxylase-oxygenase     

Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.Abbreviations OEE1, 2 and 3 oxygen evolution enhancer proteins 1, 2 and 3 - Rubisco ribulose bisphosphate carboxylase/oxygenase - PS photosystem - P21 and P30 C. reinhardtii thylakoid polypeptides 21 and 30     

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.