Enzyme-catalyzed synthesis of alkyl β-D-glycosides with crude homogenate ofSulfolobus solfataricus

Summary Crude homogenate of thermophilic archaebacteriumSulfolobus solfataricus, possessing a -glycosidase, has been used to synthesize different alkyl -D-glycosides starting from phenyl -D-glucoside, phenyl -D-galactoside and lactose as carbohydrate donors. High product yield (95% with respect to the carbohydrate donor) of octyl -D-glucoside has been obtained in a two-phase system containing 5% of water. The enantioselection for the galactosyl transfer to the secondary hydroxyl group of propane-1,2-diol is higher than that found using -galactosidase fromE. coli.     

Feedback regulation of RNA polymerase subunit synthesis after the conditional overproduction of RNA polymerase in Escherichia coli

Summary The and subunit of RNA polymerase are thought to be controlled by a translational feedback mechanism regulated by the concentration of RNA polymerase holoenzyme. To study this regulation in vivo, an inducible RNA polymerase overproduction system was developed. This system utilizes plasmids from two incompatibility groups that carry RNA polymerase subunit genes under lac promoter/operator control. When the structural genes encoding the components of core RNA polymerase (, and ) or holoenzyme (, , and ⁷⁰) are present on the plasmids, induction of the lac promoter results in a two fold increase in the concentration of functional RNA polymerase. The induction of RNA polymerase overproduction is characterized by an initial large burst of synthesis followed by a gradual decrease as the concentration of RNA polymerase increases. Overproduction of RNA polymerase in a strain carrying an electrophoretic mobility mutation in the rpoB gene results in the specific repression of synthesis off the chromosome. These results indicate that RNA polymerase feedback regulation controls synthesis in vivo.     

Characterisation of the cDNA clones of two β-tubulin genes and their expression in the potato plant (Solanum tuberosum L.)

The cDNA clones of two potato -tubulin genes were isolated from a tuberising stolon tip library. Analysis of 20 positive clones showed that they represented one or another of two different but very similar -tubulin genes, designated TUBST1 and TUBST2. The expression pattern of -tubulin genes in the potato plant was investigated by RNA blot analysis and by RT-PCR. Southern analysis of potato genomic DNA with coding and non-coding -tubulin probes revealed that there are multiple -tubulin genes in the potato genome and that there is likely to be considerable divergence in the 3 non-coding sequences. Phylogenetic analysis of plant -tubulin genes is described.     

DNA variations in regenerated plants of pea (Pisum sativum L.)

Summary The aim of this study was to determine whether DNA variations could be detected in regenerated pea plants. Two different genotypes were analyzed by cytogenetic and molecular techniques: the Dolce Provenza cultivar and the 5075 experimental line. Dolce Provenza regenerated plants showed a reduction in DNA content, particularly at the level of unique sequences and ribosomal genes. Moreover, regeneration was associated with an increase in DNA methylation of both internal and external cytosines of the CCG sequence. On the other hand, the DNA content of the 5075 line remained stable after regeneration. DNA reduction was found only in 5075 plants regenerated from callus cultures maintained for long incubation periods (about a year). The DNA variations observed are discussed both in relation to the genotype source and the role of tissue-culture stress.     

Nonspecific (“pseudo-plasmal”) dye-binding in the Feulgen nuclear stain and its blocking by azocarmin G

Summary In Feulgen nuclear staining nonspecific dye-binding due to the pseudo-plasmal reaction is intensified in isolated cells with intact cytoplasm, and cannot be eliminated by the post-irradiation method. Fluorescence intensity in the cytoplasm sometimes exceeds that of specific nuclear fluorescence, especially in brain and heart muscle cells, and it was almost impossible to perform cytofluorometric DNA quantification on such specimens. Various kinds of aldehyde-blocking agents such as sodium borohydride, 2,4-dinitrophenylhydrazine, aniline, and sodium pyrosulfite were effective in reducing the pseudo-plasmal reaction. But the blocking effects were not complete because of additional release of reactive aldehyde groups during subsequent Feulgen hydrolysis. Acidic azocarmin G produces a complete block of all pseudo-plasmal reaction in acriflavine-Feulgen nuclear staining, allowing accurate DNA-cytofluorometry to be carried out.     

Gene organization of chloroplast DNA from the broad bean Vicia faba

Summary Six protein genes have been mapped on broad bean chloroplast DNA by Southern hybridization using the tobacco chloroplast genes as probes. In broad bean chloroplast DNA, the genes for the and subunits of proton-translocating ATPase and the 32,000 dalton thylakoid membrane protein are located near the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase. The gene for the subunit of proton-translocating ATPase is distantly located from the and genes. The gene for the ribosomal protein CS19 was found close to the ribosomal RNA operon. The gene organization of broad bean chloroplast DNA is therefore quite different from that of tobacco chloroplast DNA. The nucleotide sequence of the spacer region between the large subunit and the genes of the broad bean has been determined. Conserved sequences are found among the putative promoter regions of the chloroplast protein genes.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-stranded Polydeoxyribonucleotides. 4. Topotecan Binds Preferably to the GC Base Pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption, and Raman spectroscopy. The complexes of TPT with poly(dG-dC)·poly(dG-dC), poly(dG)·poly(dC), poly(dA-dC)·poly(dG-dT), and poly(dA)·poly(dT), as well as complexes of TPT with calf thymus DNA and coliphage T4 DNA studied by us previously, have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT)·poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complex with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different composition decreases in the following order: poly(dG-dC)·poly(dG-dC) > poly(dG)·poly(dC) > poly(dA-dC)·poly(dG-dT) > poly(dA)·poly(dT). The presence of DNA has been shown to shift the monomer–dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by bridges of TPT dimers may participate in the formation of the studied type of TPT–polynucleotide complex. Molecular models of TPT complex with linear and circular supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably the whole camptothecin family) proved to represent a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Further studies on polyploid amphibians

The manner in which centromere regions of mitotic chromosomes are distributed with respect to the age of their DNA was studied. Cells of the Indian deer, Muntiacus muntjak, were grown in the presence of bromodeoxyuridine (BrdU) for two generations and stained with the fluorescent dye Hoechst 33258. Chromatids containing granddaughter DNA appear dim when compared with those containing grandparental DNA. The frequencies of the various anaphase patterns of bright and dim centromere regions were binomially distributed, indicating random distribution of chromatids with respect to the age of their DNA templates.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Transmission of plastids by pollen in Antirrhinum majus

Summary Up to now Antirrhinum was classified as a typical example for a uniparentalmaternal inheritance of the plastids. However, the findings reported here prove that also the male gametophyte of Antirrhinum may occasionally transmit plastids into the egg. This conclusion is based on genetic experiments involving a form of the plastom mutant prasinizans which is described as gelbgrüne prasinizans. In contrast to all other plastid mutations known in Antirrhinum majus this mutant originated in Sippe 50 is completely viable. In plants containing plastids of this mutant type only, the mutant character is manifested during early growth stages. Cotyledons and first foliage leaves which are initially white or white yellow, slowly turn green and become indistinguishable from normal Sippe 50. Reciprocal crosses of green Sippe 50 with gelbgrüne prasinizans gave few variegated descendants; the others were exclusively plants identical with the maternal parent as far as leaf colour is concerned (Table). The variegated individuals cannot be gene mutants since selfing and crossing experiments showed non-mendelian inheritance. Furthermore it could be ruled out that in the cross Sippe 50 x gelbgrüne prasinizans the three variegated descendants represent spontaneous new plastom mutants because the pale tissue in these plants turned green in the same way as the paternal parent. Because of the typical greening of this mutant and since plastid mutations could be ruled out we have to conclude that plastids were transmitted by the pollen parent into the egg. There these plastids multiplied together with the maternal plastids giving rise to the chimeras after sorting-out of the two plastid types. This interpretation is supported by the observation of mixed cells in tissues where the leaf variegation is finely mosaiced. The results were possible only because the plastids of the pollen parent can be unequivocally recognised.     

Peripheral Tγ lymphocyte population in head and neck cancer

Summary Peripheral T lymphocytes were measured in head and neck cancer patients and controls. The percentage was significantly higher in the 59 cancer patients than in the 46 normal controls (P<0.001). The 12 patients with recurrent disease had elevated percentages of T lymphocytes compared with the untreated group (n=31; P<0.05) and the treated, disease-free group (n=16; P<0.05). Moreover, the percentage of T lymphocytes was significantly higher in the 31 patients with regional lymph node metastasis than in the node-negative group (n=28; P<0.05). In a total of 37 patients with squamous cell carcinoma histologically graded I, II, and III, the absolute counts and percentages of T lymphocytes in the grade I group (n=13) showed significant decreases compared with those in the grade III group (P<0.05; n=6). Moreover, postoperative serial determinations of the percentage of T lymphocytes in the 14 treated, disease-free patients revealed a gradual decrease of T lymphocytes, whereas the five patients with recurrent disease had a tendency to increases in the percentage of T lymphocytes. Abbreviations used in this paper: SMF, sodium metrizoate-Ficoll; PBS, phosphate-buffered saline; SRBC, sheep erythrocytes; FCS, fetal calf serum; Hepes, hydroxyethylpiperazin elthene-sulfonic acid; E, erythrocyte; EAC, erythrocyte-antibody-complement; K, antibody-dependent killer; NK, natural killer     

The influence of mutations upon the synthesis of RNA polymerase subunits in Escherichia coli cells

Summary The influence of mutations in structural genes of and subunits of RNA polymerase upon the synthesis of these subunits in E. coli cells have been investigated. An amber-mutation ts22 in the subunit gene decreases the intracellular concentration of this subunit and the rate of its synthesis. At the same time the concentration and the rate of subunit synthesis is increased. These suggest the compensatory activation of the RNA polymerase operon that takes place under the conditions of shortage of one of the subunits. Reversions, as well as more effective supression of ts22 amber mutation, achieved by streptomycin addition, substitution of su2 by su1, or by specific mutations, result in a rise of and drop of subunit concentration and synthesis in ts22 mutant. TsX missense-mutation in the subunit gene alters the properties of the enzyme increasing, at the same time, the concentration and the rate of synthesis of both and subunits, particularly at a nonpermissive temperature. This points to an inversely proportional relationship between the rate of synthesis of RNA polymerase subunits and the total intracellular activity of the enzyme. Extra subunits are rapidly degraded in ts22 and tsX mutants.The whole complex of our data and those of others suggest that the regulation of the synthesis of RNA polymerase subunits is accomplished by interaction of a negative and a positive mechanisms of regulation which include not only activators and repressors but the enzyme itself as well.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Novel cross-strand three-purine stack of the highly conserved 5′-GA/AAG-5′ internal loop at the 3′-end termini of Parvovirus Genomes

We have used two-dimensional nuclear magnetic resonance (2D-NMR), distance geometry (DG) and molecular dynamics / energy minimization (MD/EM) methods to study a 2×3 asymmetric internal loop structure of the highly conserved `5-(GA)/(AAG)-5 bubble' present at the 3-end hairpin of the single-stranded DNA genome of parvoviruses. This motif contains an unpaired adenosine stacked between two bracketed sheared GA pairs. However, the phenomenal cross-strand G-G and A-A stacking in the tandem sheared GA pairs has undergone considerable change. A novel three-purine stacking pattern is observed instead; the inserted A18 base is completely un-stacked from its neighboring G17 and A19 bases, but well stacked with the cross-strand A4 and G3 bases to form a novel A4/A18/G3 stack that is different from the double G/G, A/A or quadruple G/G/G/G stack present in the 5-(GA)/(AG)-5 or 5-(GGA)/(AGG)-5 motifs. Unlike the bulged purine residue that usually causes about 20 degree kink in the helical axis of the parent helix when bracketed by canonical GC or AT base pairs, no significant kink is observed in the present helix containing a bulged-adenine that is bracketed by sheared G A pairs. The phosphodiesters connecting G3-A4 and G17-A18 residues adopt unusual torsional angles close to the trans domain, yet that connecting A18-A19 residues resumes the normal (g <sup>–</sup>) value. The well structured `5-(GAA)/(AG)-5' internal loop in the parvovirus genomes explains its resistance to single-strand specific endonuclease susceptibility.     

Genetic heterogeneity of propionic acidemia: Analysis of 15 Japanese patients

Summary Propionic acidemia is an autosomal recessive metabolic disease resulting from a deficiency of propionyl CoA carboxylase (PCC) activity. We have analyzed the molecular heterogeneity of Japanese propionic acidemia patients using anti-human PCC antiserum and cDNA clones coding for the two protein subunits ( and ) of the enzyme. The steady state levels of both and subunits of PCC from 15 Japanese patients were determined by Western blot. Three patients had neither nor subunits, and the amounts of both and subunits were low in 3 other patients. According to our previous data, we classified these 6 patients as having subunit deficiency. In the remaining 8 patients, subunits were normal, but the subunits were aberrant. Two patients had low levels of normal-sized subunits and 6 had subunits smaller than normal in size and greatly reduced in quantity. These 8 patients were assigned to the subunit deficiency category. One patient had apparently normal and subunits. We could not determine this patient's primary defect. These data reveal the genetic heterogeneity of molecular defects causing propionic acidemia in the Japanese. Southern blot analysis did not reveal any gross alteration in gene structure when DNA was digested withHindIII,EcoRI andTaqI. However, DNA from 3 -subunit-deficient patients, when digested withMspI and probed with PCC cDNA, revealed a unique 2.7-kb band not observed in blots of DNA from any other patient or 15 normal controls. We conclude that this alteredMspI restriction map is the result of a mutation in the subunit gene of these patients.     

The effect of the ribosomal protein S1 from Escherichia coli on the synthesis in vitro of bacterial-, DNA phage- and RNA phage proteins.

Summary Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 fromEscherichia coli on the synthesis of the coat protein of the RNA-containing phages Q and MS2, on that of an early and a ate enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicated that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.     

Evolutionary Role of Iron in Metabolism of Prokaryotes and in Biogeochemical Processes

The paper reviews data summarizing points of view about the conceptual role of iron in the appearance and evolutionary formation of the Earth and its biosphere. Participation of iron and its compounds in the appearance and development of processes of anaero- and aerobiosis as fundamental blocks of metabolism is presented as a hierarchical scheme. Magnetically arrayed iron compounds, in which the element is both in the Fe(II) and in the Fe(III) state, are considered a connecting link between the hierarchical levels. It is shown that the energy transformation Fe(II) Fe(III) is an oxidation–reduction energy core of the most important metabolic iron complexes and of processes of biogenesis both at the cellular level and in biogeosystems.