Excitation energy transfer between β-carotene and chlorophyll-a in various systems

The excitation energy transfer from -carotene to chlorophyll-a in several seminatural systems such as liposomes, lipid layers and PSI complex has been studied at room and liquid nitrogen temperature. Only in a case of PSI complex an efficient energy transfer (about 30%) from -carotene to chlorophyll-a has been observed. The results of energy transfer were discussed on the ground of Dexter's mechanism by taking into account the recently discovered energy level (¹Ag) of -carotene.Abbreviations chl-a chlorophyll-a - -car -carotene - R_DA mean donor-acceptor distance - PSI photosystem I - _exe excitation wavelength - _e emission wavelength - d optical pathlength     

Molecular cloning of cDNAs for auxin-induced mRNAs and developmental expression of the auxin-inducible genes

By differential hybridization, two auxin-inducible cDNA clones (SAR1 and SAR2) have been isolated from a cDNA library constructed to poly(A)⁺ mRNA from auxin-treated strawberry receptacles. Both the clones have been used as probes to study the expression of the auxin-induced genes in pollinated and unpollinated fruits of various stages of development and in different organs. A high level of auxin-induced mRNAs is found in pollinated fruits as compared to unpollinated fruits of the same age, suggesting that the expression of the auxin-induced genes is developmentally regulated and the level of auxin-induced mRNAs is regulated by endogenous auxin. Furthermore, our data on the expression of SAR1 and SAR2 genes in pollinated and unpollinated fruits revealed a positive correlation between growth of strawberry fruit and the induction of mRNA corresponding to the SAR1 and SAR2 clones. Ethylene has no effect on the expression of the auxin-induced mRNAs. SAR1 mRNA is not detected in other parts of strawberry plants whereas SAR2 mRNA is present in roots. Furthermore, mRNA corresponding to SAR1 and SAR2 is not detected in other auxin-responsive plant systems such as pea epicotyls and bean explants.     

Selection for rate of development and pupal weight in ‘Cnephasia’ jactatana: A genetic tool for quality improvement

Selection for fast and slow rate of pupation over three successive generations in Cnephasia jactatana (Walker) (Lepidoptera: Tortricidae) produced a fast strain that pupated 2 days earlier than the slow strain. Selection for pupal weight was asymmetrical with the light selection having more effect than the heavy selection. The larval critical weight (L_CW), the larval maximum weight (L_MW) and the latent feeding period were not affected by any of the selections. Slow and light selections resulted in longer pre-pupal periods than fast, heavy or the laboratory population. Fecundity was reduced in females from slow and light selections. Selection in insects is discussed as a possible genetic tool for overall quality improvement.Résumé La sélection pour la rapidité d'apparition des chrysalides de C. jactatana Walker produit en 3 générations une lignée rapide qui se nymphose 2 jours avant la lente. La sélection pour le poids des chrysalides est asymétrique, ayant plus d'effet sur la sélection développement lent que sur la sélection développement rapide. Le poids critique larvaire (L_CW), le poids larvaire maximal (L_MW) et la période latente d'alimentation ne sont pas modifiés par ces sélections. Les lignées lente et légère ont des périodes prénymphales plus longues que les lignées rapide et lourde, ou la souche de laboratoire. Les fécondités des lignées lente et légère sont réduites. Les sélections associées des insectes sont examinées comme outil génétique potentiel permettant d'améliorer les performances globales.     

Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA

Summary The purified bacteriophage replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of O and one molecule of P. The O-P oligomer is active in the in vitro replication of ori-containing DNA.Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the replication proteins and ori DNA. It was found that the P protein binds specifically to ori-containing plasmid DNA only in the presence of O protein. About 100 molecules of O and 10 molecules of P form a complex with the ori DNA. The DNA-O-P complex was shown to be active in an in vitro replication system.Since the physical interactions between ori and O and between P and the Escherichia coli dnaB replication protein are well documented, the evidence for a O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables to direct the host replication machinery to the replication of its own DNA.     

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis

Summary A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A phage library was first constructed by ligating a Sau3A (GATC) partial DNA digest of the entire E. coli chromosome into the BamHI (G GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRB1) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: 3, 4, 5, 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of 4 using different restriction fragments. Plasmids pGL444 and pBS105 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement a dnaGts chromosomal marker at nonpermissive 40° C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.     

Comparative susceptibility and possible detoxification mechanisms for selected miticides in banks grass mite and two-spotted spider mite (Acari: Tetranychidae)

The susceptibility and possible detoxification mechanisms of the Banks grass mite (BGM), Oligonychus pratensis (Banks), and the two-spotted spider mite (TSM), Tetranychus urticae Koch, to selected miticides were evaluated with and without synergists. BGM was 112-fold more susceptible to the organophosphate dimethoate, and 24-fold more susceptible to both the pyrethroids bifenthrin and -cyhalothrin than TSM. The synergist triphenyl phosphate (TPP) enhanced the toxicities of bifenthrin and -cyhalothrin against BGM by 3.0- and 4.2-fold, respectively, and enhanced the toxicities of bifenthrin, -cyhalothrin, and dimethoate against TSM by 6.2-, 1.9-, and 1.7-fold, respectively. The synergist diethyl maleate (DEM) enhanced the toxicities of bifenthrin and -cyhalothrin against BGM by 2.2- and 2.9- fold, respectively, and enhanced the toxicity of bifenthrin against TSM by 4.1-fold. On the other hand, the synergist piperonyl butoxide (PBO) increased the toxicities of bifenthrin and -cyhalothrin by 6.0- and 2.6-fold, respectively, against BGM, and by 4.5- and 1.9-fold, respectively, against TSM. The significant synergism with these pyrethroids of all three tested synergists (except for DEM with -cyhalothrin against TSM) suggests that esterases, glutathione S-transferases, and cytochrome P450 monooxygenases all play important roles in their detoxification. However, the toxicity of dimethoate was not enhanced by these synergists in either mite species (except for TPP against TSM). Apparently, these metabolic enzymes play less of a role in detoxification of this organophosphate in these mites.     

An improved selection method for λlacZ − phages based on galactose sensitivity

The determination of thelacZ mutant frequency in gt10lacZ phage vectors isolated from the transgenic mouse strain 40.6 (MutaMouse), requires the screening of large numbers of phages on -galactosidase activity. Existing methods rely on distinguishing a few white plaques on X-gal containing plates amongst a multide of blue ones which is both time-consuming and expensive. The new screening method described here employs the galactose sensitiveEscherichia coli C lacZ recA galE strain into which a multicopy plasmid has been introduced, which results in over-expression of thegalK andgalT genes. In the presence of phenyl--d-galactopyranoside, a substrate for -galactosidase, this leads to the suppression of lacZ ⁺ phage propagation without affecting the ability of lacZ ^– phages to form plaques. With this method it is possible to screen 1.5×10⁶ phages on a single 9-cm Petri dish. Furthermore, the need for blue/white screening has been eliminated.     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Localization of 5′-nucleotidase in bovine brain myelin fraction and myelin subfractions

Purified myelin from fresh calf brain white matter was subfractionated in a discontinuous sucrose gradient; significant recovery of protein and 2,3-cyclic nucleotide 3-phosphohydrolase (CNP) and 5-nucleotidase (5N) activities occurred in all three obtained subfractions, the highest recovery being in the light subfraction; highest 5N and CNP specific activities were in medium myelin. Purified myelin was also subfractionated in a continuous sucrose gradient, with a similar localization of protein; CNP activity and 5N activity maxima suggest that myelin may be a predominant locus of 5N in bovine brain white matter. Freezing of brain white matter caused an increase in protein and in CNP and 5N total activity recoveries in denser myelin subfractions. Cytochemistry showed the reaction product of 5N in the whole myelin fraction to be associated with the innermost, outermost and medial compact myelin layers. Effects of non-ionic detergent (Lubrol WX) on 5N activity were studied, and the results also suggest the intrinsic nature of 5N as an ectoenzyme in myelin membranes. Lubrol WX was viewed as an advisable detergent for the stimulation of myelin 5N activity, but not for the solubilization of this enzyme.     

Induction of Somatic Embryogenesis and Plant Regeneration of ‘Ataulfo’ Mango (Mangifera indica)

Nucellar explants from the immature fruit of Ataulfo mango, 3–4 cm long, were used. The process had a very low efficiency (<10%); however, Ataulfo was more embryogenic than other cultivars such as Tommy Atkins or Haden in our hands. Overall, the process from nucellus to plantlet stage took 5 months. Twelve regenerated plants were obtained and are currently growing in the greenhouse. They appear to be normal and free of abnormalities. Some biochemical and molecular tests are currently being carried out.     

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.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3 extended DNAs, with the following sequence: 5-GTTAGGCTT-3 3-CAATCCGAA-5. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3 extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5 extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

Construction in vitro of "phage-plasmid" chimerae: a new tool to analyse the mechanism of plasmid maintenance.

Summary In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Ap^r plasmid: pSC138 (Timmis et al., 1975). F recombinants were shown to be chimerae between the and the F replicons. By genetical tests, we have demonstrated that both and F replication mechanisms are functional: the F recombinant behaves as a non defective plaque forming phage on sensitive bacteria and establishes itself as a stable plasmid on recA F^- homoimmune bacteria. In the extra-chromosomal state, the F recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed.     

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat

Summary Long-range physical maps of the small multigene family of the malt -amylase genes (-Amy-1) located on the long arms of wheat chromosomes 6A (the -Amy-A1 locus) and 6B (-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an -Amy-1 cDNA probe and four gene-specific genomic probes from the -Amy-B1 locus, the size of the -Amy-B1 locus was estimated to be about 700 kb and of the -Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of CpG islands are apparent in -Amy-B1, and three in -Amy-A1. Correlation between recombination frequency and physical distance within the -Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

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).     

Molecular genetic analysis of the Ta 25H deletion: Evidence for additional deleted loci

Seventeen linking clones sublocalized to the central region of the mouse X Chromosome (Chr) were screened against genomic DNA from male mice carrying the tabby-25H (Ta ^25H ) deletion. Two of these linking clones, EM131 and EM169, were found to be deleted in Ta ^25H /Y animals. Genetic mapping through Mus musculus domesticus/Mus spretus interspecific backcross progeny, segregating for the original tabby (Ta) gene mutation, was utilized to order these markers and to define nearest flanking markers to the Ta ^25H deletion (EM140 and EM171). The size of the Ta ^25H deletion was thus estimated as up to 4.5 centiMorgans (cM). The order of markers, proximal to distal, was found to be EM140/EM131, mouse androgen receptor gene (Ar)/EM169, Ta/EM171. A putative CpG-rich island and a highly evolutionarily conserved DNA probe were isolated from the DXCrc169 locus which co-segregates with the Ta locus in this study.