Spectral characteristics of phytochrome in vivo and in vitro

The absorption maximum of the far-red absorbing form of phytochrome in the difference spectrum for phototransformation (Pfr max) was investigated in vivo and in in vitro pellets from dark grown Hordeum vulgare L. primary leaves. Exposure of pellets in Honda medium from tissue pre-irradiated with red light to far red light gave a Pfr max of 734 nm, a slightly longer wavelength than was seen in vivo (730 nm). After incubation as the red absorbing form of phytochrome (Pr) for 2 h at 0° C irradiation with red light showed that Pfr max had shifted to shorter wavelength (716 nm) in Honda medium. Further incubation as Pfr for 2 h at 0° C and irradiation with far red light showed that Pfr max had shifted to longer wavelength (726 nm). Similar shifts were also seen in other media, although the peak positions were different. Phytochrome remained pelletable throughout these experiments and Pfr max is compared to that of soluble phytochrome in similar media. The results are interpreted as indicating changes in molecular environment of the putative phytochrome membrane receptor site and that Pfr max can be used to probe the nature of this binding.Abbreviations D Dark - EDTA Ethylene diamine tetra-acetic acid - F far red light - MOPS N-morpholino-3-propane-sulphonic acid - P Phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - Pfr max wavelength maximum of Pfr absorbance in a phototransformation difference spectrum - R red light     

Intraspecific variation of spectral sensitivity in threespine stickleback (Gasterosteus aculeatus) from different photic regimes

To examine the influence of the spectral characteristics of underwater light on spectral sensitivity of the ON and OFF visual pathways, compound action potential recordings were made from retinal ganglion cells of threespine stickleback from different photic regimes. In fish from a red-shifted photic regime (P₅₀ 680 nm for downwelling light at 1m), peak sensitivity of both the ON and OFF pathways was limited to long wavelength light (_max 600–620). In contrast, the ON pathway of fish from a comparatively blue-shifted (P₅₀ 566 nm) photic regime exhibited sensitivity to medium (_max 540–560) and long (_max 600 nm) wavelengths, while the OFF pathway exhibited peak sensitivity to only medium (_max 540 nm) wavelength light. In a third population, where the the ambient light is moderately red-shifted (P₅₀ 629 nm), the ON pathway once again exhibited only a long wavelength sensitivity peak at 620 nm, while the OFF pathway exhibited sensitivity to both medium (_max 560 nm) and long (_max 600–620 nm) wavelength light. These findings suggest that the photic environment plays an integral role in shaping spectral sensitivity of the ON and OFF pathways.     

Relationship between the grain pattern in the wood,domain pattern and pattern of growth activity in the storeyed cambium of trees

Summary The relationship between the arrangement of cell events occurring in cambium in a definite configuration and the grain pattern of wood was investigated. Taking into consideration the growth activity of fusiform cell ends, a model of a migrating morphogenetic wave determining an event configuration was made. Waves of length =1 m for the periods T=2 years and T=3 years and waves of lengths =l m and =0.04 m for the period T=10 years were considered. On the model, events from successive annual rings, conventionally comprising 10 cell layers each, were summed. In this way, event maps were obtained. For wave =4 mm, the domain pattern on the modelled map was compatible with the grain pattern. The domain pattern for the wave =1 m was impossible to recreate because the wave migrated too fast. In this case, the pattern of event configuration, incompatible with the grain pattern, formed microareas, which were not domains.     

Projecting population-level response of purple sea urchins to lead contamination for an estuarine ecological risk assessment

As part of an ecological risk assessment casestudy at the Portsmouth Naval Shipyard (PNS), Kittery,Maine, USA, the population level effects of leadexposure to purple sea urchin, Arbaciapunctulata, were investigated using a stage-classifiedmatrix population model. The model divided the lifehistory of A. punctulata into five classes,incorporating both, the developmental stages of thisspecies and the endpoints from a laboratory bioassay. Finite population growth rate () was themetric relating population level impact to leadexposure. An inverse relationship was observed betweenlead tissue residues in A. punctulata and. Bioassay treatments which resulted insignificant impacts on fertilization success and zygoteviability did not translate into significant effects on, unless those treatments also negativelyimpacted adult survival. These results paralleled theelasticity (relative sensitivity) analysis of themodel, which indicated that was mostsensitive to adult and subadult survival and wasrelatively insensitive to fecundity, fertilizationsuccess, or zygote survival. Model results indicatedthat the environmental lead levels observed at PNSshould not pose significant ecological risk to seaurchin populations. Additionally, the model resultsindicated that impacts to the early life stagesroutinely used in toxicity testing do not necessarilytranslate directly into impacts at the populationlevel.     

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.     

Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda

Summary Fragments of the E. coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector. The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA. Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each. Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical. The size of these dnaB gene fragments were further delimited by deletion analysis.In E. coli groPB534 in which wild-type and A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids. On the contrary, in E. coli groPB612, which is temperature-sensitive for its groP character, replication of and A is abolished at 30° C if the strain contains the groPB612 recombinant plasmid. On the other hand, replication of B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid. The results suggest that within the cell not only the quality but also the relative amounts of dnaB and P protein are crucial for phage replication.     

Temporal shifts in visual pigment absorbance in the retina of Pacific salmon

The visual pigments and photoreceptor types in the retinas of three species of Pacific salmon (coho, chum, and chinook) were examined using microspectrophotometry and histological sections for light microscopy. All three species had four cone visual pigments with maximum absorbance in the UV (_max: 357–382 nm), blue (_max: 431–446 nm), green (_max: 490–553 nm) and red (_max: 548–607 nm) parts of the spectrum, and a rod visual pigment with _max: 504–531 nm. The youngest fish (yolk-sac alevins) did not have blue visual pigment, but only UV pigment in the single cones. Older juveniles (smolts) had predominantly single cones with blue visual pigment. Coho and chinook smolts (>1 year old) switched from a vitamin A₁- to a vitamin A₂-dominated retina during the spring, while the retina of chum smolts and that of the younger alevin-to-parr coho did not. Adult spawners caught during the Fall had vitamin A₂-dominated retinas. The central retina of all species had three types of double cones (large, medium and small). The small double cones were situated toward the ventral retina and had lower red visual pigment _max than that of medium and large double cones, which were found more dorsally. Temperature affected visual pigment _max during smoltification.     

Spectral regions in which aquatic insects see reflected polarized light

For diverse water insects (species of Hydrophilidae, Hadraenidae, Dytiscidae, Haliplidae and aquatic Heteroptera), the attractiveness of an artificial water surface was found to vary when the polarization of the reflected light, the property by which these insects identify water, was abolished in different regions of the spectrum. The sensitivity maxima of their reflection-polarization visual systems (max(POL)) thus determined were in various spectral regions, between < 360 nm (UV) and ca. 550 nm (yellow-green). Species with max(POL) at the short-wavelength end of the spectrum would be able to identify bodies of water by polarization regardless of whether the subsurface reflection was bright or dark; nevertheless, this group includes forms that avoid water with a bright subsurface because of the intensity of the reflected light. Species with max(POL) in the long-wavelength region fail to use certain bodies of water with a bright subsurface as habitats because the light they reflect at the longer wavelengths is insufficiently polarized. That the POL system of a species has a large max could affect habitat choice; on the other hand, it could also be that systems operating in the long-wavelength region were produced in the course of adaptation to the light conditions in or above the habitat.Abbreviations max(POL) spectral sensitivity maximum for polarization vision     

Transient color sensitivity of the hill reaction during the disintegration of chloroplasts

Summary During the disintegration of isolated chloroplasts, the declining capacity for evolving oxygen in the light becomes temporarily color sensitive. The decline of Hill reaction rates follows a different time course if measured in either blue (380<<500 nm) or red light (>600 nm). Later, when only one-third or less of the original maximal capacity of the particular preparation is left, the rates of oxygen evolution in red or blue light become the same again.These studies were supported by grant No. NGR-10-004-018 from the U.S. National Aeronautics and Space Administration.     

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.     

Molecular cloning of the T4 genome: organization and expression of the tail fiber gene cluster 34--38

Summary T4 derivatives that carry T4 tail fiber genes 34–38 have been isolated and characterized by genetic, structural and functional analysis. 32 T4 recombinants were identified by a marker rescue screen of 310 T4 clones generated by restriction of partial cytosine-containing T4 DNA with either HindIII or EcoRI and ligation into appropriately cleaved vectors. These tests defined 15 recombinant classes with respect to the contiguous stretches of genome recovered. Restriction enzyme structural analysis identified 7 HindIII fragments and 7 EcoRI fragments, established a restriction map covering about 11 kb, and indicated the orientation of the DNA inserts within the vectors. The cloned tail fiber genes are expressed efficiently from promoters and complement in vivo T4 phage carrying amber mutations in the tail fiber genes. Polypeptides corresponding to gp34-gp38 have been detected by SDS polyacrylamide gel electrophoresis of ³⁵S-labeled extracts of appropriate T4 recombinant infected UV-treated host cells. The genetic, structural and functional maps of the T4 tail fiber gene cluster have been correlated, and provide a rational approach to genetically directed DNA sequence analysis of genes 34–38 and their mutant variants that affect the assembly, structure and function of the tail fibers.     

Spectral sensitivity of visual cells of the compound eye ofBlatta orientalis

Responses of single visual cells of the anterior part of the compound eye of the oriental cockroachBlatta orientalis were recorded intracellularly. Two spectral types of cells were discovered: ultraviolet receptors with _max 361 nm and green-sensitive receptors with _max 503 nm. The spectral curve of the whole eye, measured by the electroretinogram, included two peaks (=350–370 and 500 nm) and a minimum between 400 and 430 nm. This last fact is interpreted as additional evidence of the dichromatic vision of the cockroach.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 57–61, January–February, 1985.     

The glutamine synthetase gene family of Arabidopsis thaliana: light-regulation and differential expression in leaves, roots and seeds.

Summary Glutamine synthetase (GS) plays an important role in the assimilation of nitrogen by higher plants. We present here a molecular analysis of the GS polypeptides, mRNAs, and genes of Arabidopsis thaliana. Western blot analysis of leaf and root protein extracts revealed at least two distinct GS polypeptides; 43 kDa and 39 kDa GS polypeptides were present in leaves, while only a 39 kDa GS was detected in roots. The 43 kDa GS polypeptide is light-inducible. In etiolated seedlings only the 39 kDa GS was detected. However, upon greening the 43 kDa GS increased to levels comparable to those observed in light-grown plants. Four distinct GS cDNA clones, Atgsl1, Atgsrl, Atgsr2 and Atk6 were isolated and characterized. Their complete nucleotide and deduced amino acid sequences are presented. The coding sequences of the four clones are 70–88% similar while their 5 and 3 untranslated regions exhibit less than 50% similarity. Northern blots of leaf, root and germinated seed RNA revealed that the four cDNAs hybridize to mRNAs which are differentially expressed in the organs of Arabidopsis thaliana. Atgsl1 is leaf-specific and hybridizes to a 1.6 kb mRNA. Both Atgsr1 and Atgskb6 hybridize to 1.4 kb mRNAs which are expressed in both roots and germinated seeds. Atgsr2 hybridizes to a 1.4 kb mRNA, which is primarily expressed in roots with low levels of expression in seeds and leaves. Atgsl1, which represents the leaf-specific mRNA, is induced by light. Atgsl1 mRNA levels increase during the greening of etiolated seedlings while Atgsr1 levels remain constant. Southern blot analysis indicated that the Arabidopsis genome contains at least four and possibly five distinct GS genes.     

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.     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.     

Localization of the metJBLF gene cluster of Escherichia coli in lambda met transducing phage

Summary The position of the metJBLF gene cluster in the transducing phage dmet102 was determined by ligation of its leftmost EcoRI fragment (102-1) to the BCDEF (nin5) EcoRI fragment of gtl (BC) and characterization of the resultant recombinant phage. The new transducing phage carries about 6kb of bacterial DNA which contains the entire met gene cluster including the promoter of its rightmost member metF. Reasonable estimates of the coding capacity required for the four genes indicate that most of the bacterial DNA of the recombinant phage is occupied by the met gene cluster.     

Studies on lambda virulent mutants

Summary Lambda virC mutant, presumably an operator mutant for the operon including x, y, CII and O genes (Fig. 1), produce clearish plaque on a sensitive bacteria.Four revertants producing turbid plaques were isolated from virC and the mutational sites of which were studied. One (tw ₁) is located very close to and on the left side of virC₃₄, and another (tw ₃₂) is at the almost same site of virC₃₄. The others (tx ₆ and tx ₅₃) are located on the right side of virC₃₄. tx recombinants have been isolated and characterized. These recombinants produce very turbid plaques and the rate of the repressor formation in the presence of CIts repressor is somewhat higher than that of wild type. tx develops very poorly after infection to sensitive cells but CItx develops normally. tx lysogens synthesize two to three times more exonuclease than the wild type lysogen. On a function of x region for the repressor formation and on a presence of a possible anti-repressor were discussed. The mutant tw ₁ might be a promotor mutation of the CI-rex operon.This material has been published as an abstract in Jap. J. Genetics 45, 474 (1970).     

Host specificity of DNA produced by Escherichia coli. XVI. Phage lambda DNA carries a single site of affinity for A-specific restriction and modification

Summary Bacteria with A-specific restriction plate unmodified phage with an efficiency of 10^-2. One mutational event can produce restriction insensitive (sA^o) mutants of . These differ from the original sA form of by no other property than their response to A-host specificity. Two-parental phage crosses involving sA and sA^o, respectively, as non-selective marker allowed to map sA between genes cII and O. These data indicate that sA is the only site on DNA with affinity for A-specific restriction. DNA is thus an interesting substrate in in vitro A-specific restriction and modification. Using an assay based on the infectivity of DNA on helper-infected bacteria, A-specific modification activity was found in partially purified sonicates of bacteria with A-host specificity. In parallel to modification, ³H-methyl label from s-adenosylmethionine, the only cofactor required for modification, was transferred to unmodified DNA. No association of radioactivity was observed in control experiments with DNA from either modified ·A or from asA^o mutant. These data suggest that A-specific modification is brought about by DNA methylation and that the sA^o mutation not only abolished the affinity for A-specific restriction, but also for A-specific modification.