Marker Lines Research Articles

外反母しにおける種子骨回旋角度の検討

The position of the hallucal sesamoid needs to be included in an evaluation of hallux valgus. In order to quantify the rotational of position of the hallucal sesamoids from weightbearing tangential radiograph, a new tangential positioning device was established. A lead marker line was placed on the most depression aspect of this device, and fitt to the metatarsal head resion. This marker line showed the horizontal plane, and the sesamoid rotation angle (SRA) was measured. The SRA consisted of the angle between tangential line of the most inferior aspect of mediallateral sesamoids and marker line. Hallux valgus angle (HVA) was measured from anteroposterior radiograph. Measurements were made of 20 feet in 10 patients with hallux valgus and 20 feet in 10 normal subjects. A comparison between the two groups showed a significant difference with respect to the SRA. There was a high correlation between SRA and HVA (r=0.876). This device can result in reliable radiographs, and the rotational position of the sesamoids can be assessed quantitatively. We concluded that the SRA is reliable and useful for assessing the rotational position of sesamoids in hallux valgus.

Fourier transform infrared investigation of non-heme Fe(III) and Fe(II) decomposition of artemisinin and of a simplified trioxane alcohol.

Fourier transform infrared spectra are reported for the Fe(III)- and Fe(II)-mediated activation of the antimalarial agents artemisinin 1 and its simplified synthetic analogue, trioxane alcohol 2. By monitoring the frequencies of the newly established marker lines in the FTIR spectra, the products of the Fe(II) and Fe(III) reactions have been characterized. In both reactions, artemisinin is activated giving a product mixture of a ring-contracted tetrahydrofuran acetatal 3, C(4)-hydroxy deoxyartemisinin 4, and deoxyartemisinin 5. These data illustrate that the oxidation state of the iron places no restrictions on the endoperoxide reduction mechanism. The FTIR difference (light - dark) spectra indicate that the endoperoxide moiety of artemisinin is photolabile and that the resulted products have the same vibrational characteristics as those observed in the reactions with Fe(II) and Fe(III). The use of 18O-18O enriched endoperoxide in 2 has allowed us to identify two oxygen sensitive modes in the reactions with Fe(II). The reduction of the peroxide bond by Fe(II) in trioxane alcohol 2 follows both the C-C cleavage and 1,5-H shift pathways and produces a ring-contracted tetrahydrofuran acetal 6 which is converted to tetrahydrofuran aldehyde 7 and C(4)-hydroxy deoxytrioxane alcohol 8, respectively. The cleavage of the O-O bond in 1 and 2 by iron and the ability to correlate vibrational properties of the reaction products with structural properties of the isolated products suggest that infrared spectroscopy is an appropriate tool to study the mode of action of antimalarial endoperoxides.

Vibrational Analysis of Metalloporphyrins with Electron-Withdrawing NO2 Substituents at Different Meso Positions

The structure of metalloporphyrins is to a major extent determined by its peripheral substituents. To explore the influence of NO2 meso substituents, we have measured polarized resonance Raman spectra of Ni(II)(5-NO2−octaethylporphyrin) and Ni(II)(5,15-NO2−octaethylporphyrin) at various excitation wavelengths in the Soret and Q band region in CS2. We obtained very complex and overcrowded spectra that were self-consistently analyzed by a global fitting procedure. This revealed that numerous Raman lines in the Raman frequency region are composed of sublines. This includes all prominent structural marker lines. Thus, we found that for both substances at least three conformers coexist in solution. The resonance excitation profiles of the corresponding sublines indicate that these conformers exhibit different degrees of nonplanarity. Altogether, the data suggest that the nitro groups destabilize the porphyrin macrocycle and increase conformational heterogeneity. We also performed a normal-mode calculation with...

Cellular dislocations: experiment and modeling

The concept of cellular dislocations (CDs) as topological defects in a regular grain array has previously been used as a convenient analogy with regular dislocations for modeling of deformation and grain growth processes on the scale of grain groups. This work provides experimental evidence supporting the existence of CDs. Results of observations of deformation relief at the surface of superplastically deformed samples under different strain states, distortion of marker lines due to grain group sliding, and in situ scanning electron microscope (SEM) deformation are provided. These data can be interpreted in terms of CDs. Finite element modeling (FEM) of sliding of grain groups indicates that stresses and strain are localized within a two grain size around CD.

An immortalized jimpy oligodendrocyte cell line: defects in cell cycle and cAMP pathway.

Normal and jimpy oligodendrocytes in secondary cultures were transfected with plasmids containing the SV40 T-antigen gene expressed under the control of the mouse metallothionein-I promoter. Two immortalized stable cell lines, a normal (158N) and jimpy (158JP) cell line, expressed transcripts and proteins of oligodendrocyte markers, including proteolipid protein (PLP), myelin basic protein (MBP), and carbonic anhydrase II (CAII). Galactocerebroside and sulfatide were also detected with immunocytochemistry. Immunoelectron microscopy using gold particles showed that the truncated endogenous jimpy PLP was distributed throughout the cytoplasm and in association with the plasma membrane of cell bodies and processes. The length of the cell cycle in the jimpy oligodendrocytes in the absence of zinc was 31 h, about a 4-h longer cell cycle than the normal line. In the presence of 100 microM zinc, the cell cycle became 3 h shorter for both cell lines, with the jimpy cell cycle duration remaining 4 h longer than the normal line. Interestingly, the jimpy cell line showed a significant deficiency in stimulation via the cAMP pathway. While the level of oligodendrocyte markers (PLP, MBP, and CAII) were significantly increased by dibutyryl cAMP (dbcAMP) treatment in the normal cell line, no changes were observed in the jimpy cell lines. This observation, together with previous results showing jimpy oligodendrocyte's failure to respond to basic fibroblast growth factor (bFGF), suggests a role for PLP in a signal transduction pathway. Jimpy and normal oligodendrocytes transfected with the SV40T antigen gene, driven by the wild-type promoter of mouse metallothionein-I, continue to express properties of oligodendrocytes and therefore provide a powerful model to explore the function of myelin proteins and to dissect the complexity of the jimpy phenotype.

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.

An Improved Diluent for Long-Term Liquid Storage of Chicken Semen

A prototype chicken semen diluent formulation has been developed based on the results of preliminary experiments. Preliminary experiments indicated the substitution of glutamate for the acetate and phosphate generally present in chicken semen diluents improved post-insemination fertility levels obtained from chicken semen following 24-96 hours of in vitro storage. Additional preliminary experiments indicated potassium could be substituted for the sodium generally contained in chicken semen diluents without affecting post-insemination fertility levels. The dual biological buffers, BES and BIS-TRIS, that replace the single biological buffer sometimes contained in chicken semen diluents have opposing effects on the diluent’s pH and permit adjustment of the prototype diluent’s pH to a range of values without altering either its osmolarity or chemical composition. Semen collected from males of the Minnesota Dominant Marker Line was diluted 1:1 in the prototype diluent and stored in vitro for 96 hours at 4°C. Groups of twenty-four hens were inseminated with the stored semen samples. Osmolarities from 265-339 mOsm did not affect post-insemination fertility levels obtained from semen stored in the prototype diluent. The proportion of fertile eggs obtained post insemination from semen stored in the prototype diluent was significantly greater than that obtained from chicken semen stored in BPSE-l in the Chi-square test. Further investigations are necessary to determine the basis for the superiority of the newly-developed poultry semen diluent formulation to BPSE-I.

Nontransitivity of sperm precedence in Drosophila.

Sperm competition is an important component of fitness in Drosophila, but we still do not have a clear understanding of the unit of selection that is relevant to sperm competition. Here we demonstrate that sperm competitive ability is not a property of the sperm haplotype, but rather of the diploid male's genotype. Then we test whether the relative sperm competitive ability of males can be ranked on a linear array or whether competitive ability instead depends on particular pairwise contests among males. Sperm precedence of six chromosome-extracted lines was tested against three different visible marker lines (cn bw, bwD, and Cy), and the rank order of the six lines differed markedly among the mutant lines. Population genetic theory has shown that departures from transitivity of sperm precedence may be important to the maintenance of polymorphism for genes that influence sperm competitive ability. The nontransitivity seen in sperm precedence should theoretically increase the opportunity for polymorphism in genes that influence this phenotype.

Cloning of regulatory sequences mediating guard-cell-specific gene expression

This report describes the use of promoter trap lines from the model plant Arabidopsis thaliana to clone regulatory sequences that mediate guard-cell-specific reporter gene expression. Stomatal guard cells represent a highly differentiated cell type within the epidermis of green tissues of higher plants. They control the stomatal aperture in response to different endogenous and environmental signals in order to optimize carbon fixation while minimizing water loss. We screened available promoter trap lines for guard-cell-specific activation of a β-glucuronidase ( uidA) reporter gene in order to obtain marker lines for guard-cell development and to gain access to regulatory pathways leading to gene expression which is restricted to this cell type. From two lines identified we successfully cloned upstream regulatory sequences. For one line, guard-cell-specific promoter activity was confirmed by re-introducing the uidA gene, fused to the newly identified regulatory sequences, into the Arabidopsis nuclear genome. However, DNA sequences downstream of the uidA/T-DNA insertion sites in the original promoter trap lines revealed no obvious coding regions in the corresponding orientation, indicating that we have probably identified ‘cryptic’ promoters, being active in guard cells.

Are the T-DNA Mutants Amenable to Standard Recombination Analysis?

Genetic analysis with T-DNA mutants often brings difficulties resulting from instability of the transgenic phenotype. In this work three different Arabidopsis thaliana T-DNA embryonic lethals and one T-DNA morphological mutant were analyzed in F2 progeny after 15 different crosses with marker lines for individual chromosomes. F2 analysis of 44 segregation ratios revealed segregation distortion of similar character consisting in abnormal excess of nontransgenic plants to the detriment of transgenic ones. We quantified this phenotypic drift (d) on the basis of phenotypic ratios given the respective formulas. The d values indicate the rate of F1 gametes which loose the T-DNA mutation or ability of its expression. The obtained d value were relatively high, 0.4 to 0.9 for individual crosses. It makes the standard recombination analysis with insertional mutants very problematic or even impossible.

Histochemical analysis of root meristem activity in Arabidopsis thaliana using a cyclin:GUS (β-glucuronidase) marker line.

We used a transgenic Arabidopsis line expressing a translational fusion between a mitotic cyclin and the reporter gene β-glucuronidase (GUS) to investigate cell divisions in postembryonic root meristems. The fusion protein contains the cyclin destruction box (CDB) and this leads to a rapid degradation of the chimeric GUS-protein after mitosis. Hence, the staining pattern of the meristem marks dividing cells. We observed that upon germination the first cell divisions occur in epidermis cells at the junction with the hypocotyl. Moreover, the accelerated root growth on media supplemented with sucrose correlates with an increased number of dividing cells and an enlargement of the root meristematic zone. The conditional root expansion mutants pom pom1 and procuste1 (quill) suppress this sugar effect leading to a smaller meristematic zone. Simultaneous visualisation of the nucleus revealed that the CYCAT1:CDB:GUS expression is subcellularly localised around the nucleus. This particular staining starts at prophase and disappears after the completion of the new cell wall. In metaphase the staining invades the cytoplasm whereas in the telophase it concentrates again around the nucleus. This cell cycle-dependent distribution was used to characterise the two root specific cytokinesis mutants pleiade1 and hyade1. In both mutants, cells which fail to develop a complete cell wall during cytokinesis divide synchronously in further cell divisions leading to multinucleate cells. These experiments demonstrate the usefulness of the CYCAT1:CDB:GUS marker line for studying cell division of wild-type and mutants. Furthermore, this line can be used to analyse the influence of biotic and abiotic signals on the rate and spatial distribution of cell divisions.

NONTRANSITIVITY OF SPERM PRECEDENCE IN DROSOPHILA

Sperm competition is an important component of fitness in Drosophila, but we still do not have a clear understanding of the unit of selection that is relevant to sperm competition. Here we demonstrate that sperm competitive ability is not a property of the sperm haplotype, but rather of the diploid male's genotype. Then we test whether the relative sperm competitive ability of males can be ranked on a linear array or whether competitive ability instead depends on particular pairwise contests among males. Sperm precedence of six chromosome-extracted lines was tested against three different visible marker lines (cn bw, bwD, and Cy), and the rank order of the six lines differed markedly among the mutant lines. Population genetic theory has shown that departures from transitivity of sperm precedence may be important to the maintenance of polymorphism for genes that influence sperm competitive ability. The nontransitivity seen in sperm precedence should theoretically increase the opportunity for polymorphism in genes that influence this phenotype.Corresponding Editor: L. Nunney

Strain, displacement and rotation associated with the formation of curvature in fold belts; the example of the Jura arc

A new simplified genetic classification scheme for arcuate fold–thrust belts is proposed. Based on total strain patterns and displacement vector fields, we distinguish three extreme end-member models: (1) `Oroclines', pure bending of an initially straight belt, (2) `Piedmont glacier' with divergent transport directions and (3) `Primary arcs'. A simple geometric model set-up for the simulation of strain patterns in primary arcs with uniform transport direction demonstrates that divergent strain trajectories and rotations of passive marker lines do not require any divergence in displacement directions. These often quoted arguments are insufficient for the identification of `Oroclinal bending' or `Piedmont glacier' type of arc formation. Only three-dimensional restorations of an arc provide the critical information about displacement directions. In their absence, arc parallel stretches and rotations in comparison with total strains provide the most useful criteria for the distinction of arc formation modes. As an example, the Jura fold–thrust belt of the external Alps is discussed. A large set of strain data includes total shortening estimates based on balanced cross-sections, local strain axes orientations from the inversion of fault populations [Homberg, C., 1996. Unpublished PhD thesis, Université de Paris VI (France)], tectonic stylolites and micro-strains from twinning in sparry calcite. Strain trajectories (maximum shortening direction) computed from these data define a strongly divergent fan with a 90° opening. A complete displacement vector field for the entire Jura has been determined from balanced cross-sections augmented with three-dimensional `block mosaic' restorations [Philippe, Y., 1995. Unpublished PhD thesis, Université de Chambéry (France)]. Displacement vectors diverge by about 40°, markedly less than strain trajectories. The non-parallelism between strain trajectories and transport directions indicates that considerable wrenching deformation did occur in both limbs of the Jura arc. Paleomagnetically determined clockwise rotations of 0–13° from ten sites (Kempf, O., et al., Terra Nova 10, 6–10) behind the right-hand half of the Jura arc and two sites with a combined 23° anticlockwise rotation behind the left-hand half of the arc are and additional argument in favor of such a wrenching deformation. We conclude that the Jura arc formed as a `Primary arc' with a minor component of `Piedmont glacier' type divergence in transport directions.

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.

Binding of the cationic 5-coordinate Zn(II)-5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin to DNA and model polynucleotides: Ionic-strength dependent intercalation in [poly(dG-dC)

The localization of the water-soluble cationic porphyrin ZnTMpyP(4) [Zn(II) derivative of 5,10,15,20-tetrakis(4-N-methylpyridyl)porphyrin] in its complex with [poly(dG-dC)]2 is studied as a function of the solution ionic strength μ (μ = 0.20–0.03). It is shown that the position of the Soret band maximum of ZnTMpyP(4) shifts from 436 to 446 nm on complexation, while its intensity markedly decreases (∼40%) when μ decreases from 0.20 to 0.03. This suggests that the porphyrin is mainly intercalated in [poly(dG-dC)]2 at μ = 0.03. Shifts of resonance Raman marker lines under ZnTMpyP(4) complexation to [poly(dG-dC)]2 at μ = 0.03 also support porphyrin intercalation. At last, a time-resolved transient absorption study of the porphyrin triplet state quenching by molecular oxygen shows that, at μ = 0.03, biexponential kinetics of triplet state quenching is observed with time constants ∼12 μs (40%) and ∼35 μs (60%), that is, much longer than that of the free ZnTMpyP(4) species (∼3 μs). Such a strong “shielding effect” of [poly(dG-dC)]2, resulting in the ∼35-μs component, is characteristic for porphyrin intercalation. The other component at ∼12 μs likely corresponds to a less protected, “partially intercalated” species in the polynucleotide. It is assumed that a decrease of the ionic strength μ (in terms of Na+ concentration) decreases the neutralization of the negative charges of the polynucleotide phosphate groups and, therefore, allows cationic ZnTMpyP(4) molecules to come closer to the nucleotides, this favoring, in a second step, full (or partial) intercalation. Since ZnTMpyP(4) is known to be axially ligated by a H2O molecule, intercalation undoubtedly must result in a loss of its axial ligand. As far as we know, this is the first observation of such an axial ligand release in metalloporphyrin, induced by intercalation in a DNA sequence. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 302–312, 1999

Stomata Patterning on the Hypocotyl ofArabidopsis thalianaIs Controlled by Genes Involved in the Control of Root Epidermis Patterning

Stomata complexes are epidermal specialized structures typical of the upper aerial part of plants (shoot). In the model plantArabidopsis thaliana,we show that in the hypocotyl (the junction between the shoot and the root), stomata are organized according to a clear pattern reminiscent of the root epidermis pattern. Although stomata complexes are typical of the shoot epidermis, their pattern on the hypocotyl is under the control of genes involved in root epidermis patterning. Moreover, we have isolated a GFP marker line for the hypocotyl epidermal cells which do not differentiate stomata complexes. In this line the root and the hypocotyl epidermal patterns are similar. Our data support the existence of interactions between developmental mechanisms involved in the control of the apical/basal polarity and the radial symmetry of the plant body.

An improved method for direct gene transfer and subsequent regeneration ofArabidopsis thaliana Landsbergerecta and two marker lines

A protocol for PEG-mediated transformation of protoplasts is described forA. thaliana ecotype Landsbergerecta and two marker lines derived from it, M4 and M10. The optimal transformation conditions were: 14 µg plasmid DNA and 28 µg carrier DNA per 6 x 105 protoplasts in 15% (w/v) PEG solution. Based on the hygromycin resistance conferred by the transgene, relative transformation frequencies of 2.5–3.2% and absolute transformation frequencies of 1–2 x 10−4, were obtained. Shoot regeneration frequencies of 40–60% were achieved, and fertile transgenic plants of the three tested lines were obtained. Southern blot hybridizations demonstrated multi-copy integration patterns in most cases. Hygromycin resistance segregation patterns of 3:1 and 15:1 were found, as well as unexpected segregation patterns, suggesting that modifications in gene expression took place and that these can progressively occur over successive generations.

Telosomic Mapping of Four Genetic Markers in Durum Wheat

Few morphological or physiological genes that are useful as genetic markers have been mapped in durum wheat (Triticum turgidum L.). Our objective was to use aneuploid stocks to map four recessive genes in durum wheat. The genes included cn-A1d in the chlorina mutant CDd6, cn-B1a in the chlorina mutant CDd1, cc in a chocolate-brown chaff (CBC) stock and bh in a supernumerary spikelet stock. Previous studies had located cn-A1d to 7AL, cn-B1a to 7BL, bh to 2AS, and cc to an unknown arm of chromosome 7B. We observed independent segregation of cc and cn-B1a in the F2 and F3 of CBC/CDd1. Segregation for chaff color among disomic F2 plants of Langdon dimonotelosomic 7BS/CBC-CDd1 indicated that cc was in 7BS. Parental marker lines were crossed to appropriate Langdon dimonotelosomic or double ditelosomic plants. We crossed double-telotrisomic (13'' + t1t'') F1 plants to the parental marker line and classified the testcross plants for chromosome number and marker phenotypes. The map distance of each gene from the centromere was 46.6 +/- 3.8 cM for cn-A1d in 7AL 42.6 +/- 4.3 cM for cn-B1a in 7BL, 33.5 +/- 4.1 cM for cc in 7BS, and 8.5 +/- 2.1 cM for bh in 2AS.

KTP laser-resistant properties of the reinforced laryngeal mask airway

We have assessed, in vitro, the effect of KTP laser strike on the reinforced laryngeal mask airway (RLMA) under a variety of conditions. At power densities normally encountered in clinical practice, using a divergent KTP laser beam, the RLMA could not be penetrated and did not ignite with laser strike. The RLMA was penetrated at a high power density of 6.94 W mm-2 after 45-60 s. A flame appeared over the RLMA shaft at this power density after 12-35 s. The black marker line on the RLMA shaft was somewhat more vulnerable to the effects of laser strike. The flow of oxygen and nitrous oxide within the shaft did not appreciably alter the laser-resistant properties of the RLMA. The RLMA cuff was more vulnerable to laser strike than was the shaft and was penetrated at very low power densities. Filling the cuff with saline had a protective effect and penetration did not occur at power densities which caused penetration of air-filled cuffs (0.37 W mm-2).

Organization and cell differentiation in lateral roots of Arabidopsis thaliana.

Lateral root formation in plants involves the stimulation of mature pericycle cells to proliferate and redifferentiate to create a new organ. The simple organization of the root of Arabidopsis thaliana allows the development of lateral root primordia to be characterized histologically. We have divided the process of lateral root development into 8 stages defined by specific anatomical characteristics and cell divisions. To identify the cell types in the developing primordium we have generated a collection of marker lines that express beta-glucuronidase in a tissue- or cell type-specific manner in the root. Using these tools we have constructed a model describing the lineage of each cell type in the lateral root. These studies show that organization and cell differentiation in the lateral root primordia precede the appearance of a lateral root meristem, with differential gene expression apparent after the first set of divisions of the pericycle.