Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum

Summary The nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5-and 3-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Scherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identity with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA.gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5- and 3-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.     

Impact of the Regulatory Regions of Retrotransposon copia on the Level of Its Expression in Testes of Drosophila melanogaster

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5 long terminal repeat (LTR) and the 5 untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3 end of LTR increased lacZ expression in the testes, whereas deletion of the 5 end of LTR increased it. This indicated that a positive regulator ofcopia expression is at the 5 end of LTR and that negative regulators are at the 3 end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.     

Formation of P1, P2-dinucleoside 5′-pyrophosphates under potentially prebiological conditions

Summary Organic pyrophosphates such as UppA and NAD are formed when a solution containing a nucleotide, a nucleoside 5-polyphosphate, Mg²⁺ and imidazole are allowed to dry out. We suggest that this synthesis may have occured concurrently with oligonucleotide formation.Abbreviations Im Imidazole - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - U uridine - p_nA adenosine 5-poly-phosphate containing n phosphate residues - pU uridine 5-phosphate - AppA P₁,P₂-diadenosine 5-pyrophosphate - UppA P₁-(uridine 5)-P₂-(adenosine 5)-pyrophosphate - ImpA adenosine 5-phosphorimidazolide - NMN nicotinamide mononucleotide - NAD nicotinamide-adenine dinucleotide     

Physiological factors determining formation of plastocyanin and plastidic cytochrome c-553 in Scenedesmus

Physiological conditions necessary for the formation of plastocyanin and the concurrent cessation of cytochrome c-553 formation were studied in cells of copper-deficient Scenedesmus acutus after the addition of copper. Plastocyanin is formed after a lag-phase, leaving constant the content of plastidic cytochrome c-553. Therefore, the concentration of plastocyanin per cell increases and the concentration of cytochrome c-553 decreases during growth. Formation of plastocyanin during the induction period studied is dependent on light intensity. In the dark, there is a 90% inhibition, whereas under light intensities above 50 Wm^-2, a ratio of 1.3 molecules plastocyanin per 1,000 molecules chlorophyll is attained.Plastocyanin formations is inhibited by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), but not by moderate concentrations of 3-[3,4-dichlorophenyl]-1,1-dimethylurea (DCMU), and by keeping the algae under a nitrogen atmosphere without CO₂. Concurrently, the cultures treated with FCCP show a decreased endogenous ATP level. The ATP is necessary for plastocyanin formation.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-[3,4-dichlorophenyl]-1,1-dimethylurea - pcv packed cell volume     

Flower color modifications of Torenia hybrida by cosuppression of anthocyanin biosynthesis genes

White and blue/white varieties of Torenia hybrida were successfully obtained from the blue variety cv. Summerwave (SWB) by cosuppressing expression of two of the genes involved in anthocyanin biosynthesis; chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR). Such molecular breeding is the only precise and efficient way to create flower color variation in SWB due to its male and female sterility. Flower color and the degree of suppression varied between transgenic lines, and anthocyanin biosynthesis was more consistently suppressed in the dorsal petal lobes, ventral petal lobes and corolla tube than lateral petal lobes. A pink variety was obtained by cosuppressing the flavonoid 3,5-hydroxylase (F35H) gene. Yellow torenia was obtained from T-33, an in-house cultivar that contained both carotenoids and anthocyanins, by cosuppression of CHS or DFR genes.     

A Thiococcus sp. nov. gen., its pigments and internal membrane system

Summary A Thiococcus sp. nov. gen., the first Thiorhodaceae known to contain bacteriochlorophyll b, was isolated from river mud using filtered light above 900 m as the selecting agent in enrichments. The orange-brown colour of the immotile, Gram-negative, photolithotrophic cocci is due to two new carotenoids, 3,4,3,4-tetrahydrospirilloxanthin and 3,4,3,4-tetrahydrospirilloxanthinal. The photosynthetic pigments appear to be associated with a unique internal membrane system consisting of branched membrane tubes of even diameter (approx. 450 Å) continuous with the cell membrane. The negatively stained tubes examined in the electron microscope reveal a surface fine structure of raised bodies arranged in a regular hexagonal pattern.Dedicated to Professor Dr. C. B. van Niel on the occasion of his 70th birthday.     

Identification of factor IX mutations in haemophilia B: application of polymerase chain reaction and single strand conformation analysis

Summary The molecular characterization of two haemophilia B defects, Calgary 1 and Calgary 2, was carried out using polymerase chain reaction (PCR) amplification and direct dideoxy sequencing. It had been previously shown that the Calgary 1 mutation affects the 5 TaqI restriction site of exon VIII, whereas Calgary 2 involves the loss of the 3 TaqI site of exon VIII of the factor IX gene. Sequencing data has now revealed that each of these alterations involves a C-to-T transition within a CpG dinucleotide. In each instance an arginine residue is replaced by a stop codon. These cases represent the recurrence of each particular alteration, both of which are predicted to result in the production of a truncated protein lacking a significant part of the catalytic region. A recently developed technique that reveals base substitutions as single-strand conformation polymorphisms (SSCP) was adapted for modelling in the detection of point mutations. Referred to here as single-strand conformation (SSC) analysis, this procedure, used in association with PCR, provided a reliable and sensitive system for molecular diagnosis in each of the cases presented. Computer-generated secondary structure predictions demonstrated a strong correlation with experimental results and the technique was used to screen 11 additional patients in the same region. A change detected by SSC analysis in one patient was localized to 55 base pairs, sequenced, and identified as a conservative amino acid substitution. This patient is now referred to as Calgary 3.     

Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synechocystis PCC 6803 via targeted gene recombination.

Summary The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3-truncated gene copy into the psbA-I locus. Both rbcL genes, in the psbA-I region and at the rbc locus, were non-functional; The former due to the 3 truncation, and the latter due to a deletion in the 5-region (creating a 5 truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO₂-requiring mutant (cyanorubrum). The 3 and the 5 truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L₈S₈) and R. rubrum L₂) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.     

Large DNA inversions,deletions, and TaqI site mutations in severe haemophilia A

Large DNA inversions caused by an intrachromosomal recombination between homologous regions located in intron 22 and 5 of the factor VIII gene have recently been identified in patients with severe haemophilia A. To evaluate better the prevalence of this large inversion and to estimate the overall sensitivity of the Southern blot/hybridization method we analysed the factor VIII gene of 49 unrelated patients with severe haemophilia A. All patients were screened for the inversion mutation, TaqI site mutations, and deletions. Mutations were identified in 31 (63%) patients, and comprised 24 large inversions, 4 partial deletions, and 3 point mutations. Three different haplotypes were characterised in the patients presenting the inversion mutation, confirming its independent origin. Two novel deletions are reported: a large one spanning from intron 14 to intron 22 and a deletion of 86 bp comprising the 3 region of exon 1 and 39–41 bp of intron 1. DNA sequencing of the deletion junction showed no significant homology between normal 5 and 3 sequences around the breakpoints. A novel missense mutation is also reported: CGAGGA, Arg-2209 to Gly. These results confirm that the inversion mutation is the most common cause of severe haemophilia A and indicate that the Southern blot/hybridization assay should be used as the first method for screening of mutations in severe haemophilia A.     

Histochemical method for localization of cyclic 3′, 5′-nucleotide phosphodiesterase

Summary A histochemical method has been described for demonstration of cyclic 3, 5-nucleotide phosphodiesterase in tissues. 5-AMP is formed due to splitting of substrate cyclic 3, 5-AMP by cyclic 3, 5-AMPase. The 5-AMP is split into adenosine and phosphate by the 5-nucleotidase from added snake venom. Endogenous tissue 5-nucleotidase would contribute to this activity. The phosphate was in turn visualized by conversion to the lead salt in the presence of lead acetate and finally into brownish-black lead sulphide by treatment with yellow ammonia sulphide. Control studies with and without substrate and snake venom, as well as inhibition by theophylline, indicate the test to be specific for cyclic 3, 5-AMPase.In the eye the conjunctiva, ciliary process, choroid and retina all showed strongly positive activity. In the kidney the proximal and distal tubules both ascending and descending and the loop of Henle show strongly positive activity — the rest of the elements being negative. The cardiac and skeletal muscle exhibited very little positive activity. The liver showed only mildly positive activity. The villi of the small intestine showed strongly positive activity at the apical part of the cells. Neurons showed very little positive activity in either the cerebral cortex or the cerebellum. On the other hand, the molecular layer in the cerebellum and the plexiform layer of the cerebral cortex showed strongly positive activity. The significance of these findings are briefly discussed. T. R. Shanthaveerappa — in previous publications.     

Medicarpin and maackiain 3-O-glucoside-6′-O-malonate conjugates are constitutive compounds in chickpea (Cicer arietinum L.) cell cultures

Summary The pterocarpan phytoalexin conjugates medicarpin 3-O-glucoside-6-O-malonate and maackiain 3-O-glucoside-6-O-malonate were isolated from cell suspension cultures of chickpea (Cicer arietinum L.) cultivar ILC 3279 and structurally elucidated. Both pterocarpan conjugates are constitutive metabolites of the chickpea cell cultures. Upon application of an elicitor from yeast to the cell cultures a substantial increase in the level of the phytoalexin aglycones medicarpin and maackiain was observed although a delayed but significantly higher rise of the conjugates also occurred. The significance of the pterocarpan conjugates for phytoalexin production is discussed.Abbreviations MeGM medicarpin 3-O-glucoside-6-O-malonate - MaGM maackiain 3-O-glucoside-6-O-malonate - MeG medicarpin 3-O-glucoside - MaG maackiain 3-O-glucoside - FGM formononetin 7-O-glucoside-6-O-malonate - BGM biochanin A 7-O-glucoside-6-O-malonate - IFR NADPH: 2-hydroxyisoflavone oxidoreductase - PTS pterocarpan synthase - IGT UDP-glucose: isoflavone 7-O-glucosyltransferase - IMT malonyl-coA: isoflavone 7-O-glucoside-6 -O-malonyltransferase - RT retention time - sh shoulder - d duplette - m multiplette - s singulette     

A factor-dependent sulfotransferase specific for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in the Cyanobacterium Synechococcus 6301

A sulfotransferase isolated from the Cyanobacterium Synechococcus 6301 was found to be specific for 3-phosphoadenosine-5-phosphosulfate (PAPS). The molecular weight of this transferase has been estimated on a Sephadex-G-100 column to be about 58,000. The K _m for PAPS was determined to be 20 M. The pH optimum was 8.0. The thiol dithioerythritol was needed for activity; other thiols such as glutathione, cysteine, or mercaptoethanol did not catalyze this reaction. The transferase, however, could not react directly with the thiol. A heat-stable factor was needed in this reaction. This factor was purified by conventional techniques and its molecular weight was determined on a Sephadex-G-50 column to be about 11,500. The factor showed normal Michaelis-Menten behavior toward the PAPS-sulfotransferase. It has been identified as thioredoxin. The tranferase was inhibited by 3-5-ADP and 2–5-ADP; all other adenine-containing nucleotides such as 2-AMP, 3-AMP, 5-AMP, ADP, and c-AMP did not influence this reaction.Abbreviation PAPS 3-phosphoadenosine-5-phosphosulfate     

Formation of type II F-primes from unstable Hfrs and their recA-independent conversion to other F-prime types

Summary Four E. coli Hfr strains, representing stable (Hfr Cavalli), moderately stable (AB312) and unstable (Ra-1, Ra-2) Hfr states, were used in the isolation of a series of F plasmids. Type II Fs were found to be the most prevalent F plasmid formed from all of the Hfrs, while the percentages of tra Fs increased as the stability of the Hfr increased. Two observations suggested that F formation in unstable Hfrs like Ra-2 may proceed through a type II F precursor. First, the major F products of Ra-2 are tra ⁺ type II Fs and, second, other F types (I, II) and classes (tra ⁺, tra) from Ra-2 appeared to be deletion derivatives of a larger F progenitor. By monitoring the molecular changes that occur when the Ra-2 derived type II F pWS200 is transferred from one recA host to another, we have found that all F types and classes can be generated from pWS200 in a recA-independent manner. F sequences involved in the genetic conversions of pWS200 include the oriT locus and the directly repeated junctions of F and chromosomal DNA. A model for the formation of Fs in unstable Hfrs is postulated in which a tra ⁺ type II F primary excision product is seen to be modified, through recA-independent processes, to other F types and classes. This model differs from the current model of F formation in that independent excision events from the Hfr chromosome are not seen as the source of type I and type II Fs.These studies have also shown that the formation of tra Fs is a recA-independent process that can occur from the F and Hfr states, that -mediated deletions in pWS200 often demonstrate regional specificity in having endpoints near the ilv operon and that genetic alterations in either replication origin of pWS200 (F oriV, chromosomal oriC) stabilize the replication of this mini-Hfr cointegrate.     

Two novel isoneolacto-undecaglycosylceramides carrying Galα1→3Lewisx on the 6-linked antenna and N-acetylneuraminic acidα2→3 or Galactose α1→3 on the 3-linked antenna, expressed in porcine kidney

Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Gal13Gal, anti-type 2 lactosamine and anti-Lewisx antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI3(Gal)2-iso-nLc8Cer, and two were novel structures differing by the number of Gal3Lewisx determinants: VI3VI3(Gal)2V3Fuc-iso-nLc8, and VI3VI3(Gal)2 V3V3(Fuc)2-iso-nLc8. The single Gal3Lewis x determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI3(NeuAc)2-iso-nLc8, and VI3NeuAcVI3Gal-iso-nLc8. A novel structure was discovered presenting a Gal3Lewisx determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI3GalV3Fuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney 3fucosyltransferase synthesizes the Gal3Lewisx determinant, acting on the 6-linked before the 3-linked Gal3neolactosamine, and appears unable to synthesize the sialosylated Lewisx determinant on neolactoseries glycolipids.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

A 6′ gentamicin acetyltransferase gene allows effective selection of tobacco transformants using kanamycin as a substrate

6 gentamicin acetyltransferases detoxify aminoglycoside antibiotics containing a 6 amino group. We tested whether a 6 gentamicin acetyltransferase gene (6 gat) of Shigella sp. is suitable as selectable gene in plant transformation using kanamycin (Km) as a substrate. A comparative transformation experiment using Nicotiana tabacum SR1 protoplasts showed that 6 gat is as effective for selection of transformants as the commonly used neomycin phosphotransferase II (nptII). In stably transformed plants we detected moderate levels of the 6 gat mRNA. An enzymatic assay was developed with which the acetyltransferase activity of the protein is easily demonstrated.     

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G (-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5 region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patients's mother and a grandmother. The different phenotypes associated with the G (-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Species diversities analyzed by density and cover in an early volcanic succession

To evaluate alpha diversities, various variables such as density, cover, volume, and weight have been used. However, density is often a distinct variable from the remaining three. To clarify differences in diversity measured by those two kinds of variables, the data collected in fourteen 2×5 m permanently-marked plots on Mount Usu, Japan, which erupted during 1977 and 1978 in growing seasons from 1983 to 1989 was analyzed, using Shannon's species diversity (H) that is represented as a result of combination of species richness and evenness (J). H and J were evaluated by density (density H and J) and cover (cover H and J). Cover H and J were significantly lower than density H and J, indicating that cover H has different characteristics from density H. Those differences are due to differences in evenness, because species richness is the same. The rank orders of species density are different from those of cover. The predominance of a few perennial herbs greatly decreases cover evenness, while seedling establishment success influences density evenness. Therefore, I propose that, during the early stages of succession on harsh environments such as volcanoes, density diversity represents seedling establishment success rate while cover diversity expresses vegetative reproduction success rate.     

The host specificity system inEscherichia coli SK

E. coli SK has its own enzyme system providing DNA host specificity which differs from the known types of specificity inE. coli K12 andE. coli B. Modification and restriction are observed when the PBVI or PBV3 phages are transferred fromE. coli SK toE. coli B or K12 (and back).A methylase has been isolated fromE. coli SK cells and partly purified. This methylase catalyzesin vitro transfer of the labelled methyl groups from S-adenosylmethionine (SAM) to DNA of both phage and tissue origin which gives rise to 5-methylcytosine (5MC) and 6-methylaminopurine (6MAP). The methylase preparations isolated from the cells at the stationary growth have proved to be 1.5–1.7 times as active as the enzyme from the cells at the logarithmic growth stage. The extract ofE. coli SK cells infected with the phage S_D cannot methylate DNAin vitro. This fact is due tode novo synthesis of the enzyme which disintegrates SAM down to 5-methylthioadenosine (5MTA) and homoserine (HS). This enzyme is not found in the cells infected with the S_D phage in the presence of chloroamphenicole. The activity of the enzyme which disintegrates SAM is the highest between the 4th and the 5th minutes of infection. Thus it may be assumed that this enzyme, most probably, is an early virus specific protein and preventsin vivo methylation of the phage DNA.