Evolutionary dynamics of leucine‐rich repeat receptor‐like kinases and related genes in plants:A phylogenomic approach

Leucine‐rich repeat(LRR) receptor‐like kinases(RLKs), evolutionarily related LRR receptor‐like proteins(RLPs) and receptor‐like cytoplasmic kinases(RLCKs) have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata(Arabidopsis), Lotus japonicus,and Medicago truncatula(Legumes), Oryza sativa ssp. japonica,and O. sativa ssp. indica(Rice), we find that LRR RLKs comprise the largest group of these LRR‐related subfamilies, while the related RLCKs represent the smallest group. In addition,comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies.Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage‐specific duplication(s) in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species‐specific duplication in the two Arabidopsis species and originated from older Arabidopsis‐specific or rosid‐specific duplications. We discuss potential pitfalls related to functional prediction for genes that have undergone frequent turnover(duplications, losses, and domain architecture changes), and conclude that prediction based on phylogenetic relationships will likely outperform that based on sequence similarity alone.     

RepSeq – A database of amino acid repeats present in lower eukaryotic pathogens

Background  

Amino acid repeat-containing proteins have a broad range of functions and their identification is of relevance to many experimental biologists. In human-infective protozoan parasites (such as the Kinetoplastid and Plasmodium species), they are implicated in immune evasion and have been shown to influence virulence and pathogenicity. RepSeq is a new database of amino acid repeat-containing proteins found in lower eukaryotic pathogens. The RepSeq database is accessed via a web-based application which also provides links to related online tools and databases for further analyses.     

Non-destructive assay systems for detection of β-glucuronidase activity in higher plants

β-glucuronidase (GUS) can be qualitatively assayed in seedlings and fully grown plants without injury or irreversible damage by short term incubations in X-gluc or by spraying 4-MUG.     

Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the -glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.     

β‐glucuronidase of family‐2 glycosyl hydrolase: A missing member in plants

Glycosyl hydrolases hydrolyze the glycosidic bond in carbohydrates or between a carbohydrate and a non‐carbohydrate moiety. β‐glucuronidase (GUS) is classified under two glycosyl hydrolase families (2 and 79) and the family‐2 β‐glucuronidase is reported in a wide range of organisms, but not in plants. The family‐79 endo-β-glucuronidase (heparanase) is reported in microorganisms, vertebrates and plants. The E. coli family‐2 β‐glucuronidase (uidA) had been successfully devised as a reporter gene in plant transformation on the basis that plants do not have homologous GUS activity. On the contrary, histochemical staining with X‐Gluc was reported in wild type (non-transgenic) plants. Data shows that, family‐2 β‐glucuronidase homologous sequence is not found in plants. Further, β‐glucuronidases of family‐2 and 79 lack appreciable sequence similarity. However, the catalytic site residues, glutamic acid and tyrosine of the family‐2 β‐glucuronidase are found to be conserved in family‐79 β‐glucuronidase of plants. This led to propose that the GUS staining reported in wild type plants is largely because of the broad substrate specificity of family‐79 β-glucuronidase on X‐Gluc and not due to the family‐2 β‐glucuronidase, as the latter has been found to be missing in plants.     

A rapid method for qualitative assay of both neomycin phosphotransferase II and β-glucuronidase activities in transgenic plants

A rapid and efficient method for assaying both NPT II and GUS activities was developed. In this method, which is modified from that of McDonnell et al. (1987), and Jefferson (1987), no sample processing procedures such as grinding and centrifugation are necessary. Cut plant tissues (leaves) or intact calli or cells expressing the genes of interest are placed in wells of a microtiter plate containing reaction mixture, and after incubation the reaction mixture is directly used for both NPT II and GUS assays. For the NPT II assay, aliquots of the reaction mixture are blotted onto Whatman P81 paper through a manifold, and the product of the reaction is detected by autoradiography. For GUS activity, aliquots or the rest of the reaction mixture are observed for fluorescent emission under a hand-held UV light or read in a fluorimeter after adding stop buffer to the reaction mixture. This method is the simplest, cheapest, and quickest assays for NPT II and GUS reported to date, and is extremely efficient and suitable for assaying small amounts of samples (as little as 0.3 mg tissue), such as in transient expression assays, or for the quick screening of large numbers of samples, such as in studies of gene inheritance in transgenic plants. In our laboratory, it has been used successfully in assaying NPT II activities for transient and stable gene expression in transformed protoplasts, calli, and leaf tissues of various transgenic plants. It has also been used for detecting both NPT II and GUS activities in transgenic rice plants, in which more than 400 samples could be assayed per day per person.     

Selection of cold‐tolerant plants for growth in soils contaminated with organics

A mixture of organic chemicals (MOC) containing equal molar amounts of benzoic acid, hexadecane, 2,2‐dimethyl 4,n‐propyl‐benzene, phenanthrene, pyrene, and either cycloheptane or cis‐decahydronaphthalene (cis‐decalin) was applied to soil at rates of 0 to 8000 mg/kg. In a plant‐screening experiment, growth responses of four legume and five nonlegume species were determined at 10 and 25°C. The MOC applied at 2000 mg/kg reduced the growth of several species without resulting in significant seedling death. At 10°C, the growth of alpine bluegrass (Poa alpina L.) in the 1000 and 2000 mg/kg treatments of soil increased by more than 185%. In a plant growth response experiment, alpine bluegrass and alfalfa (Medicago sativa L.) were grown in soil that had been contaminated at rates of 0 and 2000 mg/kg. At 14 weeks, the shoot and root dry weights of alfalfa were 97% lower in the contaminated soil, while the shoot dry weight, root dry weight, and root length of alpine bluegrass were 135,235 and 268% higher, respectively. Except for pyrene, <23% of the compounds comprising the MOC remained in the soil after 4 weeks and <5% after 14 weeks. The disappearance of the MOC was not significantly influenced by the presence of alfalfa or alpine bluegrass.     

A secondary effect of transformation in Rhizobium leguminosarum transgenic for Bacillus thuringiensis subspecies tenebrionisδ-endotoxin (cryIIIA) genes

 By introducing Bacillus thuringiensis subspecies tenebrionisδ-endotoxin genes (cryIIIA) into Rhizobium leguminosarum we have produced strains for the biological control of Sitona larvae. Comparisons between a transgenic and the parent strain show that transformation has induced changes not associated with the intended function of the transgene. Although growth rates in laboratory cultures are similar for both strains, the ability to compete for nodule occupancy is greater in the transgenic than in the non-transformed parent strain. This result demonstrates the importance of studying ecological and agronomic characters of transgenic micro-organisms that could have a bearing on the safety and success of their release into the environment, even if they are not thought to be connected with the transgenes introduced. Received: 20 April 1997 / Accepted: 2 June 1997     

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of β-glucuronidase in transgenic Brassica plants

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene -glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.     

A new amplification target for PCR–RFLP detection and identification of Chlamydiaceae species

The family Chlamydiaceae contains nine species pathogenic to humans and animals, but their routine identification is hampered by inadequate detection methods. In an attempt to find a new region for PCR detection and discrimination of the Chlamydiaceae species, the 3 end of the omp2 gene of Chlamydiaceae has been examined. Since sequence data for this part of the genes of Chlamydophila felis and Chlamydia suis had not been available, the near full length of the omp2 genes of these species were cloned and sequenced. Consensus primers enabling amplification of a previously untargeted region spanning 1,030 bp at the 3 end of the gene were designed. Discrimination of all nine Chlamydiaceae species was achieved via RFLP analysis of the amplicons with RsaI and HinfI or RsaI and TaqI endonucleases or via electrophoretic mobility analysis of the RsaI restriction fragments in agarose gel with bisbenzimide-PEG. Intraspecies uniformity of the RFLP patterns was evaluated by the typing of reference strains, isolates of human and animal origin from culture collections, and clinical specimens, and by computer analysis of GenBank sequences. The 3 end of the omp2 gene was shown to be an appropriate marker region suitable for rapid identification of Chlamydiaceae species and can be used for characterization of collection strains and new isolates in taxonomic, epidemiological, and clinical purposes.     

Sudan-β-d-glucuronides and their use for the histochemical localization of β-glucuronidase activity in transgenic plants

 Synthesis of five different Sudan-β-d-glucuronides (I, II, III, IV, and RedB) was performed by condensation of a set of red Sudan diazo dyes with methyl (1-deoxy-2,3,4-tri-O-acetyl-1-trichloroacetimidoyl-α-d-glucopyran)uronate. After the acid and alcohol groups had been deprotected, the resulting compounds were used for histochemical localization of β-glucuronidase (GUS) activity in transgenic plants (Petunia hybrida, Arabidopsis thaliana, and Nicotiana tabacum) that contained the GUS reporter system. Because the cleavage of the β-glucuronide results in the liberation of an insoluble Sudan dye, Sudan substrates gave no diffusion artifacts as described for the commonly used 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc). A comparison of assays with different Sudan glucuronides and X-gluc demonstrated that the SudanIV variant is a valuable glucuronide substrate for the precise histochemical localization of GUS activity in transgenic plants. Received: 9 December 1999 / Revision received: 25 January 2000 / Accepted: 26 January 2000     

Short communication: PCR‐based detection of the polymorphism at codon 136 in the ovine prion protein gene

Abstract

In many breeds of sheep, a polymorphism at codon 136 of the prion protein gene has been shown to be strongly associated with the risk of developing scrapie. A single‐step procedure for detecting this allelic variation is described here. When performed on a series of animals, the test was in complete agreement with their genotypes as had been previously determined by sequencing. The test is potentially easier and quicker to perform than any of the variety of methods that are currently used for this purpose.     

Versatile near-infrared fluorescent probe for in vivo detection of Aβ oligomers

Amyloid-β oligomers (AβOs) enrichment in brain is highly related to Alzheimer’s pathogenesis, but tracing them in the brain by imaging technique is still a great challenge due to their heterogeneity and metastability. Herein, a new near-infrared (NIR) fluorescent probe, namely, PTO-41, was designed and synthesized to specifically target AβOs. PTO-41 possesses excellent functional properties including optimal fluorescent properties (emission maxima at 680 nm upon interacting with AβOs), high affinity (K_d = 349 nM), low cell toxicity, desirable lipophilicity (log P = 2.24), and fast wash out from the brain (brain_{2 min}/brain_{60 min} = 5.0). Furthermore, PTO-41 exhibits a high sensitivity toward AβOs in vitro phantom imaging experiments. More importantly, PTO-41 shows great capacity to differentiate between 4-month-old APP/PS1 model mice from age-matched control mice using in vivo imaging. In summary, PTO-41 almost meets all the requirements as a versatile NIR fluorescent probe for the detection of AβOs both in vitro and in vivo.     

Evolutionary dynamics of leucine-rich repeat receptor-like kinases and related genes in plants：A phylogenomic approach

Leucine-rich repeat （LRR） receptor-like kinases （RLKs）, evolutionarily related LRR receptor-like proteins （RLPs） and receptor-like cytoplasmic kinases （RLCKs） have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata （Arabidopsis）, Lotus japonicus, and Medicago truncatula （Legumes）, Oryza sativa ssp. japonica, and O. sativa ssp. indica （Rice）, we find that LRR RLKs comprise the largest group of these LRR-related subfamilies, while the related RLCKs represent the smal est group. In addition, comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies. Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage-specific duplication（s） in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species-specific duplication in the two Arabidopsis species and originated from older Arabidopsis-specific or rosid-specific duplications. We discuss＆amp;nbsp;potential pitfal s related to functional prediction for genes that have undergone frequent turnover （duplications, losses, and domain architecture changes）, and conclude that prediction based on phylogenetic relationships wil likely outperform that based on sequence similarity alone.     

Fetal genes in mother's blood: A novel mechanism for telegony?

Telegony is a discredited genetic phenomenon that a previous male may influence the characteristics of offspring subsequently borne by the same female to another male. Although its reality was acknowledged by such authorities as Charles Darwin and Herbert Spencer, it has been met with skepticism because of a lack of understanding of the theoretical basis for telegony. With the discovery of fetal genes in mother's blood, the penetration of somatic cells by sperm, and the ability of RNA to program genome rearrangement, mechanisms might exist for telegony.     

A direct solid‐phase assay specific for heavy‐metal toxicity. II. Assessment of heavy‐metal immobilization in soils and bioavailability to plants

We have used the solid‐phase MetPLA TE, an enzyme assay that is specific for heavy‐metal toxicity, to investigate metal toxicity of soils that have been amended with urban wastewater sludges or contaminated with dry deposition from metal‐plating industries. We have shown that soil toxicity, using MetPLA TE, ranged from 21 to 72.5% inhibition of enzyme activity. Evin soil, which displayed the highest toxicity, also had the highest concentrations of Pb and Zn. Metal uptake studies with ryegrass grown on Evin soil, showed Zn, Cd, and Pb accumulation in the plant that exceeds the standard levels reported for grasses

Solid‐phase MetPLA TE was also used as a tool to study the reduction of heavy‐metal toxicity following soil amendments to immobilize metals in soil and thus reduce their toxicity. It was found that the addition of 1% hydrated manganese oxide significantly reduced dissolved metals in soil, their accumulation by ryegrass, and soil toxicity as shown by MetPLA TE.     

Multiple linked β and α globin genes in Atlantic cod: A PCR based strategy of genomic exploration

Allozyme variation in Atlantic cod hemoglobins shows various signs of natural selection. We report a genomic exploration of globin genes in this non-model organism. Applying a PCR based strategy with a strict criterion of phylogenetically informative sites we estimate the number of linked β and α globin genes. We estimate PCR error rate by PCR of cloned DNA and recloning and by analysis of singleton variable sites among clones. Based on the error rate we exclude variable sites so that the remaining variation meets successively stricter criteria of doubleton and triplet variable site. Applying these criteria we find ten clusters of linked β/α globin genes in the genome of Atlantic cod. Six variable amino acid changes in both genes were found in linkage disequilibrium with silent nucleotide substitutions. A phylogenetic tree, based on our strictly phylogenetically informative sites among 57 clones from 19 individuals, is split into two major branches by an amino acid change in a β gene. This change is supported by extensive linkage disequilibrium between the amino acid change and numerous other phylogenetically informative silent nucleotide sites. The different gene sets in the genome may represent different loci encoding different globins and/or allelic variation at some loci.