Chromosomal location of genes controlling short-term and long-term somatic embryogenesis in wheat revealed by immature embryo culture of aneuploid lines

The expression of essential genes during somatic embryogenesis can be analysed by inducing aneuploid cells to undergo embryogenesis during immature embryo culture and then determining whether defects occur. Triticum aestivum disomic and aneuploid stocks, including 36 ditelosomics and 7 nullitetrasomic Chinese Spring wheats, were compared for their ability to undergo somatic embryogenesis after 2 months of in vitro immature embryo culture. Their regeneration capacity was observed after 4 and 14 months of in vitro culture to determine which chromosome arms influence the process. The large range of variation found among the tested aneuploids suggested that genetic control of the somatic tissue culture ability is polygenic. Our results indicate that genes affecting somatic embryo-genesis and regeneration are located in all of the homoeologous chromosome groups. The lack of chromosome arms 1AL (DT 1AS) and 3DL (DT 3DS) practically suppresses somatic embryogenesis, demonstrating that major genes on wheat chromosome arms 1AL and 3DL control regeneration capacity. Results suggest that plants were mainly produced from somatic embryo development. Although the control of somatic embryogenesis and regeneration is polygenic, the genes located on the long arms of homoeologous group 3 chromosomes have a major effect. We also have evidence of chromosome arms that determine the time required for regeneration.     

Characterization of the homeologous genes of C24-sterol methyltransferase in <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

Three homeologous copies of the TaSMT1 gene for C24-sterol methyltransferase, which are located on chromosomes A, B, and D of Triticum aestivum hexaploid genome, were discovered. The bioinformatic analysis of the structure of these genes and sequencing de novo promoter sequences revealed differential expression of homeologous TaSMT1 genes in leaves and roots of wheat seedlings under normal conditions and in stress.     

Genetic control of 6-phosphogluconate dehydrogenase (6-PGD) isozymes in cultivated wheat and rye

Summary The 6-phosphogluconate dehydrogenase (6-PGD) zymogram phenotypes of wheat, rye and their aneuploid derivatives were determined. Two genes involved in the production of 6-PGD isozymes were located on chromosome arms CRL (4 RL) and FRL (6 RL) of Imperial rye. On the basis of differential interactions between wheat and rye chromosomes, evidence was obtained that genes located on chromosomes 6 A, 6 BL and 7 BL control 6-PGD isozyme activities in Chinese Spring wheat. The wheat and rye 6-PGD zymogram phenotypes were indicative of homoeologous relationships between rye chromosome 6 RL to wheat chromosomes of group 6, and rye chromosome 4 RL to wheat chromosomes of group 7.     

Genome-wide discovery of missing genes in biological pathways of prokaryotes

Background

Reconstruction of biological pathways is typically done through mapping well-characterized pathways of model organisms to a target genome, through orthologous gene mapping. A limitation of such pathway-mapping approaches is that the mapped pathway models are constrained by the composition of the template pathways, e.g., some genes in a target pathway may not have corresponding genes in the template pathways, the so-called “missing gene” problem.

Methods

We present a novel pathway-expansion method for identifying additional genes that are possibly involved in a target pathway after pathway mapping, to fill holes caused by missing genes as well as to expand the mapped pathway model. The basic idea of the algorithm is to identify genes in the target genome whose homologous genes share common operons with homologs of any mapped pathway genes in some reference genome, and to add such genes to the target pathway if their functions are consistent with the cellular function of the target pathway.

Results

We have implemented this idea using a graph-theoretic approach and demonstrated the effectiveness of the algorithm on known pathways of E. coli in the KEGG database. On all KEGG pathways containing at least 5 genes, our method achieves an average of 60% positive predictive value (PPV) and the performance is increased with more seed genes added. Analysis shows that our method is highly robust.

Conclusions

An effective method is presented to find missing genes in biological pathways of prokaryotes, which achieves high prediction reliability on E. coli at a genome level. Numerous missing genes are found to be related to knwon E. coli pathways, which can be further validated through biological experiments. Overall this method is robust and can be used for functional inference.

     

Temperature-dependent Inhibition of Hypocotyl Elongation in Some Soybean Cultivars: I. Localization of Ethylene Evolution and Role of Cotyledons

Apical 2-cm hypocotyl segments from seedlings of a "short-hypocotyl"cultivar Amsoy 71, like whole seedlings, evolve about twiceas much ethylene at 25?C as at 30?C. Segments consisting oftwo cotyledons and an attached epicotyl evolve ethylene at lowrates at both 25?C and 30?C. Hypocotyl segments from seedlingsof Cutler 71 also show enhanced ethylene evolution at 25?C.Hypocotyl segments from Corsoy, a "long hypocotyl" cultivar,however, evolve ethylene at low rates at both 25?C and 30?C.Wounding of Amsoy 71 hypocotyl segments does not increase theirethylene evolution. Ethylene evolution at 25?C is reduced andthe short-hypocotyl phenomenon is reversed by partial (50%)removal of Amsoy 71 cotyledons at planting time. ¹ Journal Paper No. J-10412 of the Iowa Agriculture and HomeEconomics Experiment Station, Ames, Iowa, U.S.A. Project 2191. (Received October 5, 1981; Accepted January 28, 1982)     

豌豆蚜基因组P450基因家族的分析

细胞色素P450在各种内源和外源物质代谢中起着非常重要的作用。利用豌豆蚜Acyrthosiphon pisum基因组mRNA和氨基酸数据库研究P450基因功能的进化规律。通过生物信息学方法对豌豆蚜全基因组P450进行分析, 结果显示: 在豌豆蚜基因组中发现69个P450基因, 它们分别属于13个P450家族和18个亚家族, 是一个典型的多基因家族。进一步将这些基因与豌豆蚜ESTs数据库进行了比对分析, 其中39个候选基因有EST证据, 证明了这些P450基因在转录水平的真实性。以氨基酸相似度大于60%为标准对豌豆蚜基因组中P450基因进行分组, 69个P450基因中, 除18个基因序列因差异太大, 不能被归入任何一组, 其余51个可归入10个组, 其中8个组(包含47条序列)适合于正选择和基因转换分析。正选择和基因转换分析结果表明: 仅有1个组(含9个基因)显著受到正选择压力作用, 正选择概率大于95%的氨基酸位点分别是20T和27N, 20T位于底物识别位点SRS1, 27N位于D. helix; 有3个组(包含8个基因）显示显著的基因转换事件。 参与基因转换的基因均为CYP4家族成员, 分别是CYP4C, CYP4G和CYP4V亚家族。 参与基因转换的成员之间的蛋白相似度较高（70％~95％）, 且XM_001944991与XM_001951794同存在于SCAFFOLD12542上, XM_001945510与XM_001944057同存在于SCAFFOLD7010上。这可能暗示豌豆蚜P450基因通过基因复制, 然后通过基因转换使P450获得新的功能, 以适应多变的生存环境。此外, 鉴定出20个不同的基序, 其中有5条基序在90％以上的基因中出现。      

Subfamilies of histone H3 and H4 genes are located on most,possibly all of the chromosomes in maize

Summary It has been previously shown that in the genome of maize the multiple copies of the histone H3 and H4 multigenic families are organized into eight to ten subfamilies each containing a variable number of copies. Each subfamily is characterized by a specific proximal environment and thus can be revealed by blot-hybridization with its specific 5 probe. Restriction fragment length polymorphism (RFLP) combined with monosomic analysis was used to localize several H3 and H4 subfamilies on maize chromosomes. H3 and H4 genes were found to be located on most, possibly all of the chromosomes, revealing a remarkably dispersed organization of these multigenic families.     

Mapping of beta-tubulin genomic sequences in hexaploid oat (Arena sativa L.)

Summary The allohexaploid nature of Avena sativa L. (2n=6x=42) and the availability of aneuploid lines was exploited in designing a strategy for mapping beta-tubulin sequences in the oat genome. Evidence for a minimum of eight beta-tubulin genes was obtained by Southern-blot analysis. Three betatubulin sequences were localized to chromosomes using DNA from monosomic and nullisomic lines in the variety Sun II. One sequence was localized to the chromosome missing in nullisome I. Two other sequences were mapped to satellite chromosome 2, the chromosome that is missing in nullisome VIII and to which one ribosomal RNA gene cluster had previously been mapped. Restriction fragments carrying these two beta-tubulin genomic sequences and the cluster of ribosomal RNA sequences were missing in DNA from nullisomics VIII, IX and X, suggesting that all three nullisome classes are deficient for an identical chromosomal segment that includes these three loci. This study demonstrates how molecular analyses can be used to characterize aneuploid stocks and to better define their genetic constitution.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or the University of Minnesota and does not imply its approval to the exclusion of other products or vendors that may be suitable     

Improvements in regeneration from protoplasts of potato and studies on chromosome stability

Summary Regeneration of plants from protoplasts of potato (Solanum tuberosum) cv. Maris Bard has been achieved from four different initial culture media (ET2, ET3, CLG, VkCLG). These media differed in their hormone, salt and sugar content. Plating efficiencies were highest in the VkCLG medium, but no correlation was found between plating efficiency and regeneration frequency (i.e. the percentage of calli producing shoots). Regeneration frequencies were high on all four media; up to 95% on ET3. Chromosome counts of up to 50 regenerants selected at random from the four treatments showed no significant differences between any of the treatments, in the proportions of plants which were euploid (48), aneuploid at the tetraploid level (48±), and aneuploid with high chromosome numbers (48+ +). Highly significant differences were present, however, between shoots which rooted quickly (predominantly euploid) and those which rooted only after transfer to a rooting medium (predominantly 48+ +). Overall more than 60% of the regenerants were normal (2n=4x=48) and this is a considerable improvement on our earlier work in this cultivar (4% normal). These findings are discussed in relation to factors affecting chromosome stability. Chromosome structural rearrangements are also described.     

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar Blue Uniform was more responsive than White Uniform. Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by -glucuronidase and ELISA analyses, respectively.Abbreviations BAP 6-Benzylaminopurine - NAA -Naphthalene acetic acid - TDZ Thidiazuron - BU Blue Uniform - WU White Uniform     

Effect of alpha-isopropylmalate on the synthesis of RNA and protein in Neurospora

Summary The leu-3/-IPM (-isopropylmalate) regulatory system, previously shown to control several genes of leucine, isoleucine, valine, and histidine biosynthesis, appears likely to be involved also in the regulation of overall RNA and protein synthesis in Neurospora. Upon addition of -IPM the synthesis of all major species of stable RNA was found to be transiently inhibited by approximately 50%. A similar reduction was observed in overall protein synthesis. The inhibition was dependent in both cases on a functional leu-3 gene product, in conformance with previously established patterns of -IPM dependent gene regulation. The overt resemblance of the phenomenon described here to the stringent response of bacteria is noted but neither the mechanism of inhibition nor the precise role of -IPM in the process has been established.     

Soil temperatures and inoculation techniques affect emergence and reisolation of sclerotinia Sclerotiorum from soybean

Emergence of Amsoy soybean (Glycine max) seed inoculated withSclerotinia sclerotiorum was significantly reduced below noninoculated seed at soil temperatures of 25, 30 and 35 °C, but not at 20 °C.S. sclerotiorum was readily·reisolated from wound-inoculated stems of seedlings and nearly mature plants above the point of inoculation below to the crown area, but not from roots. The fungus was recovered from stems but not roots of seedlings grown in sterile soil for 15 days before infestation of the soil surface with a suspension of mycelium and sclerotia and assayed at 15 days after soil infestation. When compared to healthy, seed infected withS. sclerotiorum were characterized by appearing flattened.Supported in part by the Illinois Agricultural Experiment Station; Regional Project S-72; and U.S. Agency for International Development, grant csd-1922.     

Identification of nucleases related to nutrient mobilization in senescing cotyledons from French bean

The knowledge about the physiological function of plant nucleases is scarce besides that they have been involved in nucleic acid degradation related with programmed cell death processes. Cotyledons provide a suitable system to investigate this process and the changes associated to nutrient mobilization. Nuclease activities have been determined in French bean seedlings. The total nuclease activity in French bean cotyledons is lower than in embryonic axes; however, several nucleases were detected by in-gel nuclease activity assays with extracts from cotyledons of French bean and ssDNA as substrate. The nuclease activities induced during cotyledon senescence showed higher activity at neutral than at acidic pH. Five different nuclease genes belonging to S1/P1 family have been identified in French bean genome database named PVN1 to PVN5. Their relative expression in cotyledons has been determined from the start of imbibition to senescence, and three genes from this family showed expression in cotyledons. PVN1 was expressed during early stages of seedlings development, whereas PVN4 and PVN5 were expressed during cotyledons senescence. The removal of epicotyl in French bean seedlings resulted in a decrease in the activity and in the expression of the genes associated with the cotyledons senescence process, i.e. PVN4 and PVN5. At the same time, the mobilization of reserves in those cotyledons was slowed down. In the same way, the deficit in phosphate and nitrate during seedlings development led to an acceleration of induction of these genes at the same time that reserves were utilized early on the time. Therefore, the induction of PVN4 and PVN5, the two S1 nuclease genes involved in the process of cotyledon senescence, is related to nutrient mobilization, supporting a possible role for nucleic acids in nutrient recycling during cotyledon senescence.     

Cultivar differences in boron uptake and distribution in celery (Apium graveolens), tomato (Lycopersicon esculentum) and wheat (Triticum aestivum)

Species and cultivar differences in boron (B) uptake at low B availability and tolerance to high external B are known for many species but mechanisms explaining such differences remain obscure. Here we contrast B uptake and distribution between two cultivars of tomato and celery that differ significantly in their susceptibility to B deficiency. The celery cultivar S48-54-1 and tomato cultivar Brittle are known to be more susceptible to B deficiency (inefficient) than the closely related cultivars Emerson Pascal and Rutgers (efficient), respectively. B uptake and distribution was also compared in two wheat lines differing in tolerance to B excess (Chinese Spring, sensitive and Lophopyrum Amphiploid, tolerant). Results showed that there is no significant difference in either the specific uptake rate (I_M) of ¹⁰B or the relative growth rate (RGR) between the efficient cultivar (Emerson Pascal) and less efficient cultivar (S48-54-1) of celery. However, the distribution of ¹⁰B among plant organs (leaves, stems and roots) of Emerson Pascal was different from S48-54-1. In Emerson Pascal more than 63% of accumulated B was present in the shoots while in S48-54-1 only 45% of accumulated B was present in shoots. In tomato plants, in addition to differences in B distribution among plant organs between the efficient (Rutgers) and less efficient (Brittle) cultivars, the specific uptake rate of ¹⁰B was significantly higher in the efficient cultivar. In wheat, the tolerant line (Amphiploid) took up less B than the less tolerant cultivar (Chinese Spring), and the pattern of B distribution among plant organs was different with a greater percentage of B found in roots of Chinese Spring compared to Amphiploid. Differences in sensitivity to B deficiency and excess amongst cultivars and species were a consequence of either reduced B uptake as in wheat (Amphiploid), a restriction in B translocation from roots to shoot as in celery (S48-54-1) or a combination of both process as in tomato (Brittle).     

Molecular characterization of a pea β-1,3-glucanase induced by Fusarium solani and chitosan challenge

-glucanases are prominent proteins in pea endocarp tissue responding to fungal infection. We have cloned and sequenced a partial pea cDNA clone, pPIG312, corresponding to a -1,3-glucanase in pea pods challenged with the incompatible pathogen Fusarium solani f. sp. phaseoli. The insert from the partial pea cDNA was used to probe a genomic library derived from pea leaves of the same cultivar. One of the genomic clones, pPIG4-3, contained the complete coding sequence for a mature -1,3-glucanase protein. The predicted amino acid sequence of the pea -1,3-glucanase has 78% identity to bean -1,3-glucanase, 62% and 60% to two tobacco -1,3-glucanases, 57% to soybean -1,3-glucanase, 51% to barley -1,3-glucanase, and 48% to barley -1,3-1,4-glucanase. Genomic Southern analysis indicates that the pea genome contains only one -1,3-glucanase gene corresponding to the probe used in this study. Accumulation of -1,3-glucanase mRNA homologous with the pPIG312 probe was detected in pea pods within 4 to 8 h after challenge with F. solani f. sp. phaseoli, f. sp. pisi, a compatible strain, or the elicitor, chitosan. In the incompatible reaction, mRNA accumulation remained high for 48h, whereas it rapidly decreased in the compatible reaction. After fungal inoculation of whole pea seedlings, the enhanced mRNA accumulation occurred mainly in the basal region (lower stem and root). This -1,3-glucanase glucanase mRNA was constitutively expressed in the roots of pea seedlings. The sustained levels of -glucanase mRNA expression induced by the incompatible pathogen in the resistance response suggests that the enzyme contributes to the pea plant's general defense.     

Calculations of quantum tunnelling between closed and open states of sodium channels

Transitions between states of ion channels have previously been considered in terms of classical statistical mechanics. However, transitions in many systems, including some organic molecules, are known to occur by quantum mechanical tunnelling. In this report, we have calculated the time for sodium channel activation by tunnelling, starting from a mechanistic model based on the structural models of Catterall and Guy. In doing this, we have calculated the Coulomb interactions between the S4 -helix and negative residues on nearest-neighbor helices and have included longer range interactions in terms of an effective background interaction. Periodic pairing of charges between the S4 and adjacent helices in the model causes the resting and depolarized states of the channel to correspond to local minima in the S4 potential energy curve. Harmonic potentials closely fit the energy curves around each of the two minima and the energy barrier between them is closely modelled by a parabola. These approximations allow a semiclassical calculation of the S4 helix's tunnelling rate to be made. At 37°C, for an interhelix axial spacing of 10 Å, tunnelling times in the range of 1 s to a few ms were computed for a single S4 segment, depending of the equilibrium temperature of the cell membrane.     

Biodegradation of cyclic nitramines by tropical marine sediment bacteria

Undersea deposition of unexploded ordnance (UXO) constitutes a potential source of contamination of marine environments by hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX). The goal of the present study was to determine microbial degradation of RDX and HMX in a tropical marine sediment sampled from a coastal UXO field in the region of Oahu Island in Hawaii. Sediment mixed cultures growing in marine broth 2216 (21°C) anaerobically mineralized 69% or 57% (CO₂, 25 days) of the total carbon of [UL-¹⁴ C]-RDX (100 M) or [UL-¹⁴ C]-HMX (10 M), respectively. As detected by PCR-DGGE, members of -proteobacteria (Halomonas), sulfate-reducing -proteobacteria (Desulfovibrio), firmicutes (Clostridium), and fusobacterium appeared to be dominant in RDX-enrichment and/or HMX-enrichment cultures. Among 22 sediment bacterial isolates screened for RDX and HMX biodegradation activity under anaerobic conditions, 5 were positive for RDX and identified as Halomonas (HAW-OC4), Marinobacter (HAW-OC1), Pseudoalteromonas (HAW-OC2 and OC5) and Bacillus (HAW-OC6) by their 16S rRNA genes. Sediment bacteria degraded RDX to N₂O and HCHO via the intermediary formation of hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and methylenedinitramine. The present findings demonstrate that cyclic nitramine contaminants are likely to be degraded upon release from UXO into tropical marine sediment.     

Cloning and characterization of genes coding for ribosomal RNA inCampylobacter jejuni

The DNA fragments coding for ribosomal RNA inCampylobacter jejuni have been cloned from a genomic library ofC. jejuni constructed inEscherichia coli. Clones carrying DNA Sequences for rRNA were identified by hybridization of 5-end-labeled rRNA fromC. jejuni to colony blots of transformants from this gene library. Cloned DNA sequences homologous to each of 5S, 16S, and 23S rRNA were idenfified by hybridization of labeled plasmid DNA to Northern blots of rRNA. The gene coding for 23S rRNA was found to be located on a 5.5kb HindIII fragment, while the 5S and 16S rRNA genes were on HindIII fragments of 1.65 and 1.7 kb, respecitively. The DNA fragment containing the 16S rRNA gene was characterized by restriction endonuclease mapping, and the location of the 16S rRNA gene on this fragment was determined by hybridization of 5-end-labeled rRNA to restriction fragments and also by DNA sequence determination. It appears that the major portion of the coding region for 16S rRNA is located on the 1.7-kb HindIII fragment, while a small portion is carried on an adjacent HindIII fragment of 7.5 kb. Cloned rRNA genes fromC. jejuni were used to study the organization of the rDNA inC. jejuni and other members of the genùsCampylobacter.     

Characterization,genetic diversity,phylogenetic relationships,and expression of the aluminum tolerance <Emphasis Type="Italic">MATE1</Emphasis> gene in <Emphasis Type="Italic">Secale</Emphasis> species

Aluminum (Al) is the main limiting factor for crop production in acidic soils. Efflux of organic acids is one of the mechanisms that determine Al-tolerance, and an Al-activated citrate transporter (multidrug and toxic compound extrusion) MATE1 gene is involved in different species. The contribution of the rye MATE1 gene (ScMATE1) depends on the rye (Secale cereale L.) cultivars and the crosses analyzed; there is no information about different rye species. The cDNA sequences, phylogenetic relationships, Al-tolerance, citrate exudation, and expression of the ScMATE1 gene were analyzed in several cultivars and wild species/subspecies of the Secale genus. Genotypes highly tolerant to Al were found within this genus. For the first time, sequences of the cDNA of the ScMATE1 gene were isolated and characterized in wild ryes. At least two copies of this gene were found likely to be related to Al-tolerance. The sequence comparison of 13 exons of ScMATE1 revealed variability between species, but also inter- and intra-cultivars. Variations were found in the Al-induced expression of ScMATE1 gene, as well as its contribution to Al-tolerance. The pattern of citrate exudation was inducible in most of the species/subspecies studied and constitutive in few. The phylogenetic analysis indicated that ScMATE1 is orthologue of two genes (HvMATE1 and TaMATE1) involved in the Al stress response in barley and wheat, respectively, but not orthologue of SbMATE, implicated in Al-tolerance in sorghum. ScMATE1 is involved in the response to Al stress in ryes, but its contribution to Al-tolerance is complex, and like in other species, there are tolerant and sensitive alleles in the different cultivars and species studied.