Efficient regeneration system from wheat leaf base segments

Efficient plant regeneration system from leaf base segments of wheat (Triticum aestivum L.) was developed. The factors affecting the callus formation and regeneration capacity of leaf segments of two genotypes; Bobwhite and Pavon 76, were investigated. The highest number of somatic embryos (SE) was obtained on Murashige and Skoog medium supplemented with 2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid + 1 mg dm⁻³ naphthalenacetic acid (14.7 SE per segment). Highest frequency of embryogenic callus (96 %) and somatic embryo formation (24.3 SE per segment) were achieved in the first segments. The highest plantlet regeneration was obtained after transfer of embryogenic calli to regeneration medium supplemented with 1 mg dm⁻³ kinetin (6.3 plantlets per segment).     

CXII. Total synthesis of the structural gene for an alanine transfer RNA from yeast. Enzymic joining of the chemically synthesized polydeoxynucleotides to form the DNA duplex representing nucleotide sequence 1 to 20

The DNA duplex (designated [A]) corresponding to the nucleotides 1 to 20 of the major yeast alanine transfer RNA (Fig. 1) has been synthesized. The first step involved the T4 ligase-catalyzed joining of d-(5′-³²P)-C-C-G-G-A-A-T-C (segment 4, Fig. 1) to the dodecanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T (segment 1, Fig. 1), in the presence of the complementary decanucleotide d-(5′-OH)-C-C-G-G-A-C-T-C-G-T (segment 3, Fig. 1). The resulting icosanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T-C-C-G-G-A-A-T-C, was isolated free from the decanucleotide (segment 3). The synthesis of [A] was then completed by the ligase-catalyzed joining of 5′-³²P or ³³P-labeled hexanucleotide d-(5′-P)-C-C-A-C-C-A (segment 2) to the 5′-³²P or ³³P-labeled decanucleotide, d-(5′-P)-C-C-G-G-A-C-T-C-G-T (segment 3), in the presence of the above icosanucleotide.     

Observations on the oviposition behaviour ofGoniozus sensorius [Hymenoptera: Bethylidae] a parasite ofDiaphania indica [Lepidoptera: Pyralidae]

Goniozus sensorius Gordh (Hymenoptera: Bethylidae) was recorded as a major parasite of the pumpkin caterpillar,Diaphania indica (Saunders) (Lepidoptera: Pyralidae). This paper provides detailed information on the oviposition behaviour ofG. sensorius. Prior to oviposition, the parasite temporarily paralyses the host larva. The paralysed condition lasts for about 2 h. The egg laying process on each larva requires 30 to 60 min. The maximum number of eggs are laid on the 6^th and 7^th segment, and none in the terminal segments. Generally, oviposition is restricted to 1 segment of the host larva and occasionally on 3 segments. The maximum number of eggs are laid on the 2^nd and 3^rd day after emergence and the mean number of eggs laid on each host larva was 7.1.     

Hormonal control of morphogenesis in leaf segments ofCentaurium erythraea

Transversally cut leaf segments ofCentaurium erythraea were cultivated on MS medium. Effects of segment polarity, IAA and sucrose concentrations, light and medium volume on morphogenesis were studied. Shoots generally formed at lower (1.3 × 10^-6 mol 1^-1) IAA concentrations than roots and callus (1.1 × 10^-5−3.4 × 10^-5 mol 1^-1). Leaf polarity strongly modified the effect of IAA concentration, shifting organogenesis at the segment base toward decreased IAA concentrations as compared with segment apex. Light, sucrose concentration above 3 % and high medium volume changed IAA dependence of morphogenesis in various ways and generally suppressed segment polarity.     

Dark Ionic Flux and the Effects of Light in Isolated Rod Outer Segments

We have determined the permeability properties of freshly isolated frog rod outer segments by observing their osmotic behavior in a simple continuous flow apparatus. Outer segments obtained by gently shaking a retina are sensitive but nonideal osmometers; a small restoring force prevents them from shrinking or swelling quite as much as expected for ideal behavior. We find that Na⁺, Cl^-, No₃^-, glycerol, acetate, and ammonium rapidly enter the outer segment, but K⁺, SO₄⁼, and melezitose appear impermeable. The Na flux is rectified; for concentration gradients in the physiological range, 2 x 10⁹ Na⁺ ions/sec enter the outer segment, but we detect no efflux of Na⁺, under our conditions, when the gradient is reversed. Illumination of the outer segment produces a specific increase in the resistance to Na⁺ influx, but has no effect on the flux of other solutes. This light-dependent Na⁺ resistance increases linearly with the number of rhodopsin molecules bleached. We find that excitation of a single rhodopsin molecule produces a transient (~1 sec) "photoresistance" which reduces the Na⁺ influx by about 1%, thus preventing the entry of about 10⁷ Na⁺ ions. At considerably higher light levels, a stable afterimage resistance appears which reduces the Na influx by one-half when 10⁶ rhodopsin molecules are bleached per rod. We have incorporated these findings into a model for the electrophysiological characteristics of the receptor.     

栽培药材川续断地上部分体积估算的最优样方选择

以贵州省黔南州龙里县内人工栽培的川续断(Dipsacus asperoides)为研究对象, 就如何选择最优样方的面积和数目来估算川续断地上部分体积进行了研究。首先运用球缺模型计算栽培川续断的地上体积, 然后利用基于套状样方的样带调查法研究估算川续断体积时的最优样方面积, 最后利用方差法计算最优采样数目。结果表明: (1)在只考虑相对平均值和相对消耗时, 25 m × 25 m是最优样方面积; 在此基础上进一步考虑到样方的边界效应和单位面积地上体积相对平均值的变化, 得出25 m × 50 m是最优样方面积。(2)如果预计置信度1-α是0.9, 绝对误差限度d是0.12, 总体方差S²按照常规取0.25, 则25 m × 50 m对应的最佳样方数目是25。(3)该研究实际采集了25个25 m × 50 m的样方, 计算后得到整个栽培园地(面积为72696.24 m ²)川续断的总体积90%的近似置信区间为[1909.798 m³, 2214.762 m³]。     

Segmental isotope-labeling of the intrinsically disordered protein PQBP1

Polyglutamine tract-binding protein 1 (PQBP1) is an intrinsically disordered protein abundantly expressed in the brain. Mutations in the PQBP1 gene are causative for X-linked mental retardation disorders. Here, we investigated the structure of the C-terminal segment within the context of full-length PQBP1. We produced a segmentally isotope-labeled PQBP1 composed of a non-labeled segment (residues 1–219; N-segment) and a ¹³C/¹⁵N-labeled segment (residues 220–265; C-segment). Our results demonstrate that the segmental isotope-labeling combined with NMR spectroscopy is useful for detecting a very weak intra-molecular interaction in an intrinsically disordered protein.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^?) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^? transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^?. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

On the termination of the DNA replication cycle in Escherichia coli.

The initiation of the DNA replication cycle in Escherichia coli requires protein synthesis. Marunouchi &; Messer (1973) have hypothesized that an additional protein synthesis step is required for the replication of the terminal segment of the chromosome, and that replication of this segment is a prerequisite for subsequent cell division. We have not confirmed the existence of a unique terminal segment using a protocol designed to label the hypothesized segment with [³H]dThd². Our protocol avoids the increased incorporation of [³H]dThd into DNA caused by abrupt increases in temperature, a complication implicit in the technique of Marunouchi &; Messer (1973).Treatment with nalidixic acid (an inhibitor of semiconservative DNA synthesis) in sufficient concentration to prevent replication of the postulated terminal segment prevents cell division but also causes loss of viability. This makes it difficult to correlate the effect of nalidixic acid on cell division with DNA synthesis inhibition alone.     

Molecular and cytological characterization of an extra acrocentric chromosome that restores male fertility of wheat in the msH1 CMS system

A new CMS system designated as ‘msH1’ has been reported in bread wheat using the cytoplasm of H. chilense. While testing this system in different wheat backgrounds, a highly fertile line with chromosome number 42 plus an extra acrocentric chromosome was obtained. The extra chromosome did not pair with any wheat chromosome at meiosis, and progeny from this line which lack the acrocentric chromosome showed pollen abortion and male sterility. In order to establish the origin of this chromosome, FISH using H. chilense genomic DNA as probe was used and showed that it had originated from H. chilense chromosome(s). The novel chromosome did not possess sequences similar to wheat rDNA; however, the probe pSc119.2 from S. cereale containing the 120 bp family was found to occur at the end of its long arm. Data obtained from FISH and EST molecular markers confirm that the long arm of the acrocentric chromosome is indeed, the short arm of chromosome 1H^ch from H. chilense. We suggest that the novel chromosome originated from a deletion of the distal part of the long arm of chromosome 1H^ch. Neither the 1H^chS short arm, nor the whole chromosome 1H^ch restores pollen fertility of the alloplasmic wheat. Therefore, the restorer gene on the acrocentric chromosome must be located on the retained segment from the hypothetical 1H^chL, while some pollen fertility inhibitor could be present on the deleted 1H^chL distal segment. Disomic addition of the acrocentric chromosome was obtained and this line resulted fully stable and fertile.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^–) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^– transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^–. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

Structure of a Ca2 +/CaM:Kv7.4 (KCNQ4) B-Helix Complex Provides Insight into M Current Modulation

Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M currents and cardiac and auditory I_KS currents and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca^2 +/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved in both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca^2 +/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca^2 +/CaM:Kv7.4 B segment complex shows that Ca^2 +/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca^2 +/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca^2 +/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease-causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function.     

Basic and editing mechanisms underlying ion transport and regulation in NCX variants

Structure-dynamic analysis of archaeal NCX (NCX_Mj) provided new insights into the underlying mechanisms of ion selectivity, ion-coupled alternating access, ion occlusion, and transport catalysis. This knowledge is relevant, not only for prokaryotic and eukaryotic NCXs, but also for other families belonging to the superfamily of Ca²⁺/CA antiporters. In parallel with the ion transport mechanisms, the structure-dynamic determinants of regulatory CBD1 and CBD2 domains have been resolved according to which the Ca²⁺-induced allosteric signal is decoded at the two-domain interface and "secondarily" modified by a splicing segment at CBD2. The exon-dependent combinations within the splicing segment control the number of Ca²⁺ binding sites (from zero to three) at CBD2, as well as the Ca²⁺ binding affinity and Ca²⁺ off-rates at both CBDs. The exon-dependent combinations specifically rigidify the local segments at CBDs, yielding the Ca²⁺-dependent activation (through Ca²⁺ binding to CBD1) and Ca²⁺-dependent alleviation of Na⁺-induced inactivation (through Ca²⁺ binding with CBD2). The exon-dependent synergistic interactions between CBDs characteristically differ in NCX1 and NCX3, thereby underscoring the physiological relevance of structure-controlled shaping of ion-dependent regulation in tissue-specific NCX variants. How the ion-dependent regulatory modules operate in conjunction with other regulators (PIP₂, palmitoylation, XIP, among the others) of NCX is an open question that remains to be determined.     

Modulation of Cell-adhesive Activity of Fibronectin by the Alternatively Spliced EDA Segment

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA⁺ FN was significantly more potent than EDA⁻ FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA⁺ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin α5 and β1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin α5β1. In support of this conclusion, purified integrin α5β1 bound more avidly to EDA⁺ FN than to EDA⁻ FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA⁺ FN and EDA⁻ FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin α5β1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180° at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III₁₀ module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.     

P-type ATPases of eukaryotes and bacteria: Sequence analyses and construction of phylogenetic trees

The amino acid sequences of 47 P-type ATPases from several eukaryotic and bacterial kingdoms were divided into three structural segments based on individual hydropathy profiles. Each homologous segment was (1) multiply aligned and functionally evaluated, (2) statistically analyzed to determine the degrees of sequence similarity, and (3) used for the construction of parsimonious phylogenetic trees. The results show that all of the P-type ATPases analyzed comprise a single family with four major clusters correlating with their cation specificities and biological sources as follows: cluster 1: Ca²⁺-transporting ATPases; cluster 2: Na⁺- and gastric H⁺-ATPases; cluster 3: plasma membrane H⁺-translocating ATPases of plants, fungi, and lower eukaryotes; and cluster 4: all but one of the bacterial P-type ATPases (specific for K⁺, Cd²⁺, Cu²⁺ and an unknown cation). The one bacterial exception to this general pattern was the Mg²⁺-ATPase of Salmonella typhimurium, which clustered with the eukaryotic sequences. Although exceptions were noted, the similarities of the phylogenetic trees derived from the three segments analyzed led to the probability that the N-terminal segments 1 and the centrally localized segments 2 evolved from a single primordial ATPase which existed prior to the divergence of eukaryotes from prokaryotes. By contrast, the C-terminal segments 3 appear to be eukaryotic specific, are not found in similar form in any of the prokaryotic enzymes, and are not all demonstrably homologous among the eukaryotic enzymes. These C-terminal domains may therefore have either arisen after the divergence of eukaryotes from prokaryotes or exhibited more rapid sequence divergence than either segment 1 or 2, thus masking their common origin. The relative rates of evolutionary divergence for the three segments were determined to be segment 2 < segment 1 < segment 3. Correlative functional analyses of the most conserved regions of these ATPases, based on published site-specific mutagenesis data, provided preliminary evidence for their functional roles in the transport mechanism. Our studies define the structural and evolutionary relationships among the P-type ATPases. They should provide a guide for the design of future studies of structure-function relationships employing molecular genetic, biochemical, and biophysical techniques. Correspondence to: M.H. Saier, Jr.     

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.     

Fine map of the Gct1 spontaneous ovarian granulosa cell tumor locus

The spontaneous development of juvenile-onset, ovarian granulosa cell (GC) tumors in the SWR/Bm (SWR) inbred mouse strain is a model for juvenile-type GC tumors that appear in infants and young girls. GC tumor susceptibility is supported by multiple Granulosa cell tumor (Gct) loci, but the Gct1 locus on Chr 4 derived from SWR strain background is fundamental for GC tumor development and uniquely responsive to the androgenic precursor dehydroepiandrosterone (DHEA). To resolve the location of Gct1 independently from other susceptibility loci, Gct1 was isolated in a congenic strain that replaces the distal segment of Chr 4 in SWR mice with a 47 × 10⁶-bp genomic segment from the Castaneus/Ei (CAST) strain. SWR females homozygous for the CAST donor segment were confirmed to be resistant to DHEA- and testosterone-induced GC tumorigenesis, indicating successful exchange of CAST alleles (Gct1 ^CA ) for SWR alleles (Gct1 ^SW ) at this tumor susceptibility locus. A series of nested, overlapping, congenic sublines was created to fine-map Gct1 based on GC tumor susceptibility under the influence of pubertal DHEA treatment. Twelve informative lines have resolved the Gct1 locus to a 1.31 × 10⁶-bp interval on mouse Chr 4, a region orthologous to human Chr 1p36.22.