Radial transport of water across cortical sleeves of excised roots ofZea mays L.

The stationary radial volume flows across maize (Zea mays L.) root segments without steles (sleeves) were measured under isobaric conditions. The driving force of the volume flow is an osmotic difference between the internal and external compartment of the root preparations. It is generated by differences in the concentrations of sucrose, raffinose or polyethylene glycol. The flows are linear functions of the corresponding osmotic differences ( ) up to osmotic values which cause plasmolysis. The straight lines obtained pass through the origin. No asymmetry of the osmotic barrier could be detected within the range of driving forces applied ( =±0.5 MPa), corresponding to volume-flow densities of j_{v, s}=±7·10^–8 m·s^–1. Using the literature values for the reflection coefficients of sucrose and polyethylene glycol in intact roots (E. Steudle et al. (1987) Plant Physiol.84, 1220–1234), values for the sleeve hydraulic conductivity of about 1·10^–7 m·s^–1 MPa^–1 were calculated. They are of the same order of magnitude as those reported in the literature for the hydraulic conductivity of intact root segments when hydrostatic pressure is applied.Abbreviations and symbols a _s outer surface of sleeve segment - c concentration of osmotically active solute - j _{v, s} radial volume flow density across sleeve segment - Lp_s hydraulic conductivity of sleeves - Lp_r hydraulic conductivity of intact roots - _N thickness of Nernst diffusion layer - reflection coefficient of root for solute - osmotic value of bulk phase - osmotic coefficient     

Sensitive Nonradioactive Method for Screening Compounds Interacting with Neuronal α7 Cholinoreceptor

A sensitive nonradioactive method for detection of substances interacting with the neuronal 7-type nicotinic acetylcholine receptor (AChR) was proposed. The method uses biotinylated -cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with -cobratoxin (CTX) as does the whole receptor. LBED produced by heterologous expression of a gene fragment of the 7 subunit of AChR from the rat brain in Escherichia coli cells was sorbed in wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin–peroxidase complex. The ability of other compounds to interact with 7-AChR was checked according to the degree with which they inhibit the Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).     

Zur wirkung von Miracil auf menschliche Leukozytenchromosomen in vitro

Summary In human leukocyte chromosomes in vitro the thioxanthon derivative 1-(2-diethylaminoethylamino)-4-methylthioxanthone hydrochloride (Miracil D) induces especially achromatic lesions (AL) und chromatid breaks (B). The frequencies of isochromatid breaks (B) are at the control level, and chromatid translocations (RB) are only found at the two highest concentrations tested. The results are compared with the findings of Lüers (1955) on the mutagenic action of the same substance in Drosophila and with the action of the monofunctional ethyleneimino compound 2-ethyleneimino-5.6.7.8.-tetrahydronaphtoquinone-1,4 (A 137) in Drosophila and human leukocyte chromosomes in vitro. A connection of AL with mutational events seems doubtful.     

Relationships between light dependent fitness of Drosophila melanogaster and D. simulans and their genotypes for pupation site photopreference

The genetic variation in the preference for pupation sites, under conditions of varying white light intensity, was investigated in two Drosophila sibling species. In parallel, an attempt was made to examine the effect on fecundity of the level of light experienced by the flies immediately after their pupation site choice had been made. Larval photopreferences of several selection lines of Drosophila melanogaster and D. simulans were assessed in a gradient of illumination and the genetic architectures influencing this behaviour were investigated from the responses to artificial selection, reciprocal crosses between divergently-selected lines, and a chromosomal analysis. D. simulans responded only to selection for negative larval photopreference, while D. melanogaster responded strongly to selection for positive but weakly to selection for negative photopreferences. Selected lines of flies that repeatedly experienced light conditions grossly at variance with those they preferred (traumatic lines) sometimes diverged in preference from lines that experienced their chosen conditions (rewarded lines). The traumatic lines always diverged from the corresponding rewarded lines in the direction of preferences that were associated with high fitness, as revealed by low response to selection in the rewarded lines. It is suggested that for a given genotype for phototactic behaviour there is a corresponding particular light dependent fitness, as measured by female fecundity.     

Free Ia Eα chain expression in the E α + :E β − : recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E chain expression in the E ⁺ E ^– I-region recombinant strain, A.TFR5. A.TFR5 (A ^f E ^k, ap5), a recombinant between A.CA (A ^f E ^f) and A.TL (A ^k E ^k), carries the E ^k subregion. Previous results have shown that it expresses the E chain, but at reduced levels relative to E ⁺ E ⁺ strains. No E chains were detected, which is consistent with the A.TFR5E gene being derived from the A.CA parent, which carries the null E ^f allele. In this paper, the defect in E-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E, in the region of the large intervening sequence of E. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E message, but no E message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular E chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular E, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E, the E chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.     

A xyloglucan-oligosaccharide-specific α-d-xylosidase or exo-oligoxyloglucan-α-xylohydrolase from germinated nasturtium (Tropaeolum majus L.) seeds

The -xylosidase which is involved in the postgerminative mobilisation of xyloglucan in nasturtium seed cotyledons has now been purified to apparent homogeneity by a facile procedure involving lectin affinity chromatography. The purified enzyme, a glycoprotein, moved as a single band (apparent molecular weight 85000) on sodium dodecyl sulphate-gel electrophoresis, whilst isoelectric focusing gave a number of enzymatically active protein bands spanning the range pI = 5.0 to 7.1 (maximum activity at pI = 6.1). The enzyme did not hydrolyse the simple -xylosides p-nitrophenyl--d-xylopyranoside and woprimeverose (-d-Xyl(16)-d-Glc), or polymeric tamarind-seed xyloglucan. It released xylose from a complex mixture of oligosaccharides produced by exhaustive hydrolysis of tamarind seed xyloglucan using the xyloglucan-specific endo-(14)--d-glucanase from germinated nasturtium seeds (M. Edwards et al. 1986, J. Biol. Chem., 261. 9489–9494). The three xyloglucan oligosaccharides of lowest molecular size were purified from this mixture and were shown by ¹H-nuclear magnetic resonance (¹H-NMR) and enzymatic analysis to have the structures:Abbreviations Con A Concanavalin A - DEAE diethylaminoethyl - Gal galactose - Glc glucose - HPLC high-performance liquid chromatography - M _r apparent molecular mass - NMR nuclear magnetic resonance - pI isoelectric point - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Xyl xylose Much of the work reported in this paper was carried out with the aid of the European Community's Science Stimulation Action (Contract No. ST2P-0250-UK), and we wish to record our appreciation of this support.     

On the Evolution of Dopa decarboxylase (Ddc) and Drosophila Systematics

We have sequenced most of the coding region of the gene Dopa decarboxylase (Ddc) in 24 fruitfly species. The Ddc gene is quite informative about Drosophila phylogeny. Several outstanding issues in Drosophila phylogeny are resolved by analysis of the Ddc sequences alone or in combination with three other genes, Sod, Adh, and Gpdh. The three species groups, melanogaster, obscura, and willistoni, are each monophyletic and all three combined form a monophyletic group, which corresponds to the subgenus Sophophora. The Sophophora subgenus is the sister group to all other Drosophila subgenera (including some named genera, previously considered outside the Drosophila genus, namely, Scaptomyza and Zaprionus, which are therefore downgraded to the category of subgenus). The Hawaiian Drosophila and Scaptomyza are a monophyletic group, which is the sister clade to the virilis and repleta groups of the subgenus Drosophila. The subgenus Drosophila appears to be paraphyletic, although this is not definitely resolved. The two genera Scaptodrosophila and Chymomyza are older than the genus Drosophila. The data favor the hypothesis that Chymomyza is older than Scaptodrosophila, although this issue is not definitely resolved. Molecular evolution is erratic. The rates of nucleotide substitution in 3rd codon position relative to positions 1 + 2 vary from one species lineage to another and from gene to gene. Received: 2 June 1998 / Accepted: 3 September 1998     

Extent of heterosis in rice (Oryza sativa L.) under cold stress conditions — yield and its components

Summary The nature and extent of heterosis for grain yield and its component characters was estimated in a seven-parent F₁-diallel cross in rice under the cold stress conditions of Palampur, representing sub-temperate climate of the mid hills of Himachal Pradesh. The highest heterotic effects for grain yield were observed in Himalaya 1/Phul Patas 72 and China 988/Himdhan hybrids, which gave 60.36 and 32.48% heterosis, respectively, over mid parent, 38.76 and 26.86% heterosis, respectively, over high parent and 32.30 and 26.86% heterosis, respectively, over the best variety, Himdhan. Heterosis for grain yield in these crosses was due to an increase in tiller number, panicle length, spikelets/panicle and 1,000-grain weight. Large number of crosses exhibited significant heterosis for high spikelet sterility and the majority of them had significant heterosis for late flowering, taking more than 120 days to flower, which resulted in lack of heterosis for grain yield in such crosses due to cold stress at the reproductive stage.Part of the thesis submitted by the senior author to Himachal Pradesh Agricultural University, Palampur (H.P.) India, in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Agriculture     

The effect of antibiotics on the inhibition of callus induction and plant regeneration from cotyledons of sugarbeet (Beta vulgaris L.)

Callus induction and plantlet regeneration from cotyledonary expiants of sugarbeet was observed utilizing two media formulations, MS and a modified MS termed RVIM both supplemented with 1.0 g/ml BAP as the sole growth regulator. Callus induction was genotype dependent The USDA line 8787 produced the highest response for callus induction followed by Betaseed 4587 and the USDA line C600. This order was conserved on both media formulations. Shoot induction was consistently higher averaging 32% from the RVIM formulation over the 3 genotypes compared to 25% from MS. The antibiotics geneticin, gentamycin, hygromycin, kanamycin and phleomycin were screened with the modified RV system utilizing Betaseed 4587. Callus growth was inhibited by levels of 50 g/ml geneticin, 150 g/ml gentamycin, 10 g/ml hygromycin, 150 g/ml kanamycin and 20 g/ml phleomycin. The results indicate that the concentrations of antibiotics used to inhibit callus induction will be sufficient for use as selectable markers in transformation experiments with Beta vulgaris.Abbreviations B₅ basal medium (Gamborg et al, 1968) - BAP N⁶-Benzylaminopurine - IBA Indole-3-butanoic acid - MS basal medium (Murashige and Skoog 1968) - RVIM modified MS basal medium (Freytag et al, 1988) - MES (2[N-Morpholino] ethanesulfonic acid     

The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression

Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5 or 3 end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5 end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.     

Biological chromatology. The laws of colour and design in nature

Summary Biochromatology can be defined as the study of the relationships between colours, and between colours and design in nature. Certain laws concerning biochromatics can be formulated in the terminology of the Colour Circle. One of them is that planes in warm colours and planes in cold colours never adjoin. Another law is that design is never polychromatic. It seems that the observed biochromatical facts can be explained with the help of the principle optimal manifestation of colour, which in turn is based on the formative principles of matter itself.     

Nucleic acid metabolism in yeast

Summary The three haploid yeast strains T2tmp1-3, T2tmp1-1, and T6tmp1-51 auxotrophic for 5-dTMP differ in their requirement for thymidylate: 72, 16, and 3 g 5-dTMP/ml will restore optimal growth, respectively. Thymidylate low requirement in strain T2tmp1-1 and T6tmp1-51 is termed tlrA and tlrC, respectively. When the growth medium is made 5x10^-4 M for 5-dTMP only strain T6tmp1-51 is severely inhibited in RNA and DNA synthesis. This inhibition is reversible after removal of excessive 5-dTMP. The inhibitory characteristic is in marked contrast to thymineless death due to the lack of 5-dTMP in strain T6tmp1-51 where only DNA synthesis stops while RNA synthesis continues. The inhibitory effect of 5x10^-4 M 5-dTMP is not due to the 5-dTMP auxotrophy but to the thymidylate low requiring character (tlrC) in strain T6tmp1-51. The arrest of RNA and DNA synthesis by high concentrations of exogenous 5-dTMP suggests a regulatory role of either the monoor triphosphate on nucleoside or nucleotide biosynthesis in yeast.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Molecular phylogeny of the subgenus Sophophora of Drosophila derived from large subunit of ribosomal RNA sequences

RNA sequencing has been used to assess the relationships among species of the subgenus Sophophora of the genus Drosophila. Two divergent domains, D1 and D2, of the large ribosomal RNA (28S), totalling 550 nucleotides have been sequenced using the rRNA direct sequencing method. A tree has been reconstructed from the neighbor-joining algorithm and the confidence intervals were evaluated by the bootstrap procedure. Results have shown that the branching of the willistoni and saltans groups of the subgenus Sophophora is very ancient and probably predates that of the subgenus Drosophila. The other groups and subgroups of Sophophora are clustered in three main lineages: 1) the melanogaster and oriental subgroups; 2) the montium subgroup; 3) the ananassae subgroup of the melanogaster group clustered with the fima and obscura groups. Thus, in comparison with our results, several taxa of various ranks appear paraphyletic (the genus Drosophila, the subgenus Sophophora and the melanogaster group). Our biochemical phylogeny is only in partial agreement with the pattern of Throckmorton's radiations as well as with classical taxonomy, both based on morphological data.     

Polyphyly of Lordiphosa and its relationships in Drosophilinae (Diptera: Drosophilidae)

The phylogenetic relationships of Lordiphosa and some taxa in Drosophilinae were analysed on the basis of a total of forty‐one selected drosophilid species. These included eighteen species of five Lordiphosa species‐groups as the main target, twenty‐three species representative of the major drosophiline ingroup taxa and four species of Steganinae as outgroup. Sixty‐eight morphological characters of adults were subjected to cladistic analysis. From the results it is concluded that Lordiphosa is polyphyletic; the Lo. tenuicauda species‐group and genus Nesiodrosophila form a single monophyletic group; Lordiphosa proper (i.e. Lordiphosa spp. minus the tenuicauda group) comprises another monophyletic group; within Lordiphosa proper the fenestrarum, nigricolor and denticeps groups are all monophyletic, but monophyly of the miki group is not strongly supported; genera Hirtodrosophila and Scaptomyza and subgenus Sophophora are all monophyletic; and within Drosophilinae, genus Scaptodrosophila is the first to have split from the main lineage, but the branching order of other clades, Chymomyza, Lordiphosa proper, Sophophora, Hirtodrosophila, Nesiodrosophila+ Lo. tenuicauda group, Scaptomyza, Dorsilopha and subgenus Drosophila, remains unresolved. The topology of maximum parsimony cladograms suggests that Lordiphosa proper lies close to Sophophora as proposed previously, although its phylogenetic position could not be determined conclusively. By contrast, bootstrap values tended to contradict another hypothesis that Lordiphosa and Scaptomyza are sister groups.     

Monocyte-derived dendritic cells that capture dead tumor cells secrete IL-12 and TNF-α through IL-12/TNF-α/NF-κB autocrine loop

This study focused on the question of how monocyte-derived dendritic cells (Mo-DCs) that capture dead tumor cells (Mo-DCs-Tum) secrete interleukin 12 (IL-12) and tumor necrosis factor (TNF-). Mo-DCs-Tum showed higher secretions of IL-12 and TNF- than were shown by Mo-DCs. Enhanced nuclear factor-kappa B (NF-B) activation was also induced in Mo-DCs-Tum within 6 h. The NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed both IL-12 and TNF- secretions from Mo-DCs-Tum. Administration of recombinant TNF- or IL-12 enhanced IL-12 or TNF- secretion respectively in Mo-DCs-Tum. Addition of anti-TNF- or anti-IL-12 neutralizing antibody decreased NF-B activation and IL-12 or TNF- secretion in Mo-DCs-Tum. These results suggest that TNF- or IL-12 secretion induces NF-B activation, and it stimulates further TNF- and IL-12 secretions, i.e., an IL-12/TNF-/NF-B autocrine loop, in Mo-DCs-Tum. Thus, Mo-DCs-Tum secrete a large amount of IL-12 and TNF- through accelerated NF-B activation induced by the IL-12/TNF-/NF-B autocrine loop.     

Identification and chromosomal locations of aconitase gene loci in Triticeae species

Summary Two systems of monomeric aconitase (ACO) isozymes, designated ACO-1 and ACO-2, were identified in Triticum aestivum and in five diploid Triticeae species. The gene loci Aco-A1, Aco-B1, and Aco-D1 were located in T. aestivum cv. Chinese Spring chromosome arms 6Aq, 6Bq, and 6Dq, respectively, and the gene loci Aco-A2, Aco-B2, and Aco-D2 in 5 Aq, 5 Bq, and 5Dq, respectively. Aco-1 gene loci were also identified in 6E of Elytrigia elongata, 6HL of Hordeum vulgare cv. Betzes, 6RL of Secale cereale PI 252003, 6S¹ of T. longissimum, and CSU-31 of T. umbellulatum. Other Aco-2 gene loci were identified in 5RL of S. cereale cv. King II and 4EL of E. elongata. Conservation of synteny relationships is indicated among the species studied for the genes identified, with the exception of Aco-E2; the presence of this gene in 4EL suggests that E. elongata differs from Chinese Spring and King II by a translocation involving 4E and 5E.Adapted from a thesis submitted to the Graduate College, Texas A&M University, by K.J.C. in partial fulfillment of the requirements for the M.S. degree in Genetics     

Phylogenetic analysis of chloroplast restriction enzyme site mutations in the Saccharinae Griseb. subtribe of the Andropogoneae Dumort. tribe

Chloroplast (cp) DNA from 32 genotypes representing eight genera and 19 species from the Andropogoneae tribe was analyzed using 15 restriction enzymes and Southern hybridization with 12 cpDNA probes that span the complete rice chloroplast genome. Six of the genera, Saccharum, Miscanthus, Erianthus, Narenga, Eccoilopus, and Sclerostachya, are part of the Saccharinae subtribe, whereas the other two, Zea and Sorghum, were used as outgroups. Narenga, Miscanthus, Erianthus, and Sclerostachya are presumed to have been involved in the evolution of Saccharum officinarum (noble or high sucrose sugarcane) via S. spontaneum and S. robustum. Southern hybridization with the rice cpDNA probes surveyed approximately 3% of the S. officinarum Black Cheribon genome and yielded 62 restriction site mutations (18 informative) that were analyzed using cladistic parsimony and maximum likelihood. These site mutations placed the 32 genotypes into nine different chloroplast groups; seven from within the Saccharinae subtribe and the two outgroups (maize and Sorghum). Phylogenetic inferrence under various assumptions showed that the maternal lineages of Narenga, Miscanthus, Sclerostachya, and Saccharum formed a monophyletic group. This group displayed little variation. On the other hand, 5 of 6 Erianthus species and Eccoilopus longisetosus formed a separate group. The Old World Erianthus/Eccoilopus chloroplast was very different from that of the rest of the Saccharum complex members and was slightly more related to that of Sorghum bicolor. Placement of these Erianthus/Eccoilopus genotypes was, therefore, in conflict with analyses based on morphology. Surprisingly, Erianthus trinii, a New World species, had the same restriction sites as did one Miscanthus sinensis. One Miscanthus sp. from New Guinea that has a very high chromosome number (2n=192) had the same restriction sites as the majority of the Saccharum genus, suggesting that introgression between these genera occurs in the wild. The Saccharum genus was separated into two clades by single site mutation: one containing S. spontaneum, and the other containing all of the remaining Saccharum species and all 8 commerical hybrids (from various regions of the world). A physical map of the chloroplast of Saccharum officinarum Black Cheribon was constructed using 5 restriction enzymes.     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene

Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the -galactosidase expression pattern in transformed lines carrying different lengths of 5 flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5 flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5 flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5 flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5 flanking DNA is not necessary for embryonic survival and development to adult flies. Correspondence to: P.M. Salvaterra