Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.     

Abundance patterns of lily pollen cDNAs: characterization of three pollen-preferential cDNA clones

Twenty-five clones were randomly selected from a mature pollen cDNA library of Easter lily (Lilium longiflorum Thunb.) in order to study the abundance of pollen-expressed mRNAs and the functional roles of the proteins encoded by these mRNAs. Plaque hybridization experiments were conducted to estimate indirectly the expression level of the mRNAs. Based on the hybridization frequency in the mature pollen library, the cDNA clones were divided into three abundance groups. Eight clones belonged to a high abundance class in which each cDNA clone was present in the mature lily pollen library at a frequency between 0.3 and 3%. Six of these clones were not found in cDNA libraries made from carpel, leaf, or root, suggesting that they are preferentially expressed in pollen. Fourteen clones belonged to a medium abundance class and were present in the mature pollen library at a frequency between 0.01 and 0.08%. The remaining three clones, which were present at a frequency below 0.01%, were grouped as a low abundance class. Almost all of the cDNA clones which belong to either the medium or low abundance class were also detected in the leaf library. Northern blot hybridization with three of the highly abundant cDNA clones confirmed their preferential expression in anther. In situ hybridization experiment with one of the clones showed the pollen-specific expression of the clone in mature anther. DNA sequence analysis revealed that the clone LMP131 encodes a peptide which is highly homologous to the tomato pollen-preferential gene, LAT59, which encodes a putative pectate lyase. The clone LMP134 encodes a peptide that shows an extensive similarity to a variety of thioredoxins. The third clone LMP132 encodes a 182-residue protein that has no significant homology to known sequences.     

Identification of a villin-related tobacco protein as a novel cross-reactive plant allergen

In a paradigmatic approach we identified cross-reactive plant allergens for allergy diagnosis and treatment by screening of a tobacco leaf complementary DNA (cDNA) library with serum IgE from a polysensitized allergic patient. Two IgE-reactive cDNA clones were isolated which code for proteins with significant sequence similarity to the actin-binding protein, villin. Northern- and Western-blotting demonstrate expression of the villin-related allergens in pollen and somatic plant tissues. In addition, villin-related proteins were detected in several plant allergen sources (tree-, grass-, weed pollen, fruits, vegetables, nuts). A recombinant C-terminal fragment of the villin-related protein was expressed in Escherichia coli, purified and shown to react specifically with allergic patients IgE. After profilin, villin-related proteins represent another family of cytoskeletal proteins, which has been identified as cross-reactive plant allergens. They may be used for the diagnosis and treatment of patients suffering from multivalent plant allergies.     

Isolation of developmentally regulated novel genes based on sequence identity and gene expression pattern.

Based on the surmise that a variety of genes might play important roles in embryonic development and tissue differentiation, and that some of them are likely to be expressed in undifferentiated ES cells, we attempted to identify new genes from the ES cell cDNA library. The modified method of expressed sequence tags (ESTs) and the examination of the expression patterns in adult tissues and in vitro differentiated ES cells were utilized in this study. We have isolated and identified several novel cDNA clones with interesting developmental expression pattern. Among the 83 clones randomly chosen, 23 clones (27.7%) have no homology to any sequences in public databases. The rest contain limited or complete sequence homology to the previously reported mammalian genes or ESTs, yet some clones have not been previously identified in the mouse. To examine the expression profile of clones during development and differentiation, sets of slot blots were hybridized with developmental stage specific or tissue specific probes. Out of 40 novel clones tested (21 totally unknown clones and 19 unidentified clones in mouse), most of them were up- or down-regulated as differentiation proceeded, and some clones showed differentiation-stage specific expression profiles. Surprisingly, a majority of genes were also expressed in adult tissues, and some clones even revealed tissue specific expression. These results demonstrate that not only was the strategy we employed in this study quite efficient for screening novel genes, but that the information gained by such studies would also be a useful guide for further analysis of these genes. It also suggests the feasibility of this approach to explore the genomewide network of gene expression during complicated biological processes, such as embryonic development and tissue differentiation.     

The isolation of cDNA clones from cucumber (Cucumis sativus L.) floral buds coming from plants differing in sex

In this study, we found flower cDNA clones which may be connected with the development of flower sex in cucumber. Two pairs of nearly-isogenic lines: gynoecious GY3 (FFMMGG) versus hermaphrodite HGY3 (FFmmGG) and monoecious B10 (ffMMGG) versus gynoecious 2gg (ffMMgg) were used for clone isolation. To obtain differentially-expressed clones, we applied the differential screening method. 454 clones from GY3 and 478 from B10 cDNA libraries were isolated. The results of RFLP analysis with 56 cDNA clones showed no clones which cosegregated with sex in cucumber. The 28 cDNA B10 and 33 cDNA GY3 clones isolated using the differential screening method were sequenced. Some of them seem to may play a role in cell differentiation or flower development. Among the 61 identified clones, 14 show high homology to plant proteins, although of unknown function. 11 show high homology to known proteins, and the possible function of some of them is discussed. For 3 clones, no significant similarity was found. The 31 clones displayed high homology to plant cDNA in EST database. The patterns of expression of five differential cDNA clones, 35GY3, 216GY3, 47GY3, 100B10 and 157B10, were analyzed in cucumber flower buds using in situ RT-PCR. The most interesting clone is 35GY3, because of its possible role in the inhibition of the development of male specific elements in the female cucumber flower.     

Expression of differentiated functions in the developing porcine small intestine

Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from poly(A) + RNA extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.     

Differentially expressed genes and proteins upon drought acclimation in tolerant and sensitive genotypes of Coffea canephora

The aim of this study was to investigate the molecular mechanisms underlying drought acclimation in coffee plants by the identification of candidate genes (CGs) using different approaches. The first approach used the data generated during the Brazilian Coffee expressed sequence tag (EST) project to select 13 CGs by an in silico analysis (electronic northern). The second approach was based on screening macroarrays spotted with plasmid DNA (coffee ESTs) with separate hybridizations using leaf cDNA probes from drought-tolerant and susceptible clones of Coffea canephora var. Conilon, grown under different water regimes. This allowed the isolation of seven additional CGs. The third approach used two-dimensional gel electrophoresis to identify proteins displaying differential accumulation in leaves of drought-tolerant and susceptible clones of C. canephora. Six of them were characterized by MALDI-TOF-MS/MS (matrix-assisted laser desorption-time of flight-tandem mass spectrometry) and the corresponding proteins were identified. Finally, additional CGs were selected from the literature, and quantitative real-time polymerase chain reaction (qPCR) was performed to analyse the expression of all identified CGs. Altogether, >40 genes presenting differential gene expression during drought acclimation were identified, some of them showing different expression profiles between drought-tolerant and susceptible clones. Based on the obtained results, it can be concluded that factors involved a complex network of responses probably involving the abscisic signalling pathway and nitric oxide are major molecular determinants that might explain the better efficiency in controlling stomata closure and transpiration displayed by drought-tolerant clones of C. canephora.     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Characterization of pollen polygalacturonase encoded by several cDNA clones in maize

A full-length cDNA clone, named PG1, abundantly expressed in late stages of pollen development, has been isolated from a cDNA library using a differential screening method with cDNA probes representative of microspores at early or late developmental stages. The encoded 410 amino acid polypeptide has significant homology with various polygalacturonases (PG) described elsewhere. Two polypeptides, of 49 and 53 kDa respectively, have been identified in the active PG fraction, isolated from mature pollen by immuno-cross-reaction with tomato PG antibodies. According to their N-terminal sequence, they can be identified as being mature peptides encoded by the PG1 cDNA clone. We propose that these two proteins derive from a unique precursor through several post-translational events, including the excision of a 22 amino-terminal signal peptide and glycosylation. PG-encoding genes form a small genomic family. Sequence analysis of three PG cDNA clones shows that they are closely related. The divergence of nucleotides between these three cDNA clones is 1%. They encode the same product.     

Analysis of sequences from human brain cDNA gene bank which are functionally active in nervous tissue and tumor cells

Construction of a human cortex cDNA bank is described as well as the isolation from this bank of pBH71 and pBH3 clones with preferential expression in nervous and in tumor cells. The clones can be included into the third class of cDNA according to Sutcliff's classification. The mRNA corresponding to this cDNA class is considered to play the key role in determination of specificity of nervous tissue. Expression of the pBH71 sequence was revealed in human cortex and in tissues of different genesis (from neuroblastoma to uterus myoma), a 2 kb mRNA which corresponds to one and the same cDNA chain having been found in all tissues under analysis. The nucleotide sequence of cDNA insertion into the pBH71 clone of 447 n.p. was determined, and particular features of cDNA nucleotide composition and possible schemes of its translation were analysed. Weak homology was found between the 3'-end of cDNA insertion of the pBH71 clone and the 3'-end region of human proopiomelanocortine. The cDNA of the pBH3 clone hybridizes with the 0.8 kb mRNA revealed in human cortex and neuroendocrine tumors of different nature. No homology was revealed between the cDNA sequence of the pBH3 clone and any genes deciphered.     

Further characterization of genes expressed during Ciona intestinalis metamorphosis

Metamorphosis of ascidians is a dynamic event by which a nonfeeding, mobile tadpole larva is transformed into a filter-feeding, fixed juvenile. This process usually begins with the settlement of the larva and is followed by a series of coordinated morphogenetic movements that rearrange organs, tissues, and cells. To identify genes that are involved in the initiation of metamorphosis, we conducted differential screening between mRNAs of swimming larvae and those of juveniles in Ciona intestinalis. This screening permitted the isolation of cDNA clones for genes whose expression is upregulated during metamorphosis, and the characterization of four such genes (Ci-meta3, Ci-meta4, Ci-meta5 and Ci-meta6) is reported here. Ci-meta3 encodes a protein with a domain found in Sp1a and the RYanodine receptor. This gene is not expressed in early swimming larvae but is expressed in the endoderm region and part of the retractile tail region in metamorphosing juveniles. The predicted proteins encoded by Ci-meta4, Ci-meta5 and Ci-meta6 do not contain any known consensus motifs, nor do they show any similarity to known proteins. Ci-meta4 and Ci-meta5 are expressed weakly in mesenchyme cells of the early larva and strongly in the metamorphosing juvenile, while Ci-meta6 is expressed in the mesenchyme in the late larva. In addition, we characterized 53 independent cDNA clones whose expression was downregulated during the period from early swimming larvae to metamorphosing juveniles by taking advantage of the Ciona intestinalis cDNA project database and BLAST searches. The expression patterns of some of these clones were changed during the larval period.     

Identificaton and characterization of stamen- and tapetum-specific genes from tomato

Summary Differential screening of a tomato cDNA library produced from pre-anthesis stamens resulted in the isolation of 25 cDNA clones that hybridized to probes made from stamen RNA and showed no hybridization to probes made from RNA of vegetative organs. The 25 clones were found to represent 11 noncross-hybridizing classes. The majority of these clones were derived from genes that were single or low copy in the tomato genome. Northern RNA blotting experiments of vegetative and floral organs at several stages of development demonstrated that expression in all 11 classes was confined to floral organs. Of the 11 classes 9 were found to be expressed exclusively in stamens prior to anthesis. Two classes showed expression in immature stamens and in petals, with one of these two additionally being expressed in mature stamens at anthesis. Clones from three of the classes that were expressed exclusively in stamens were used as probes for in situ localization of RNA in floral organs. These experiments demonstrated that expression of the genes corresponding to these clones was confined to the tapetal cells of the anthers. Expression of one of the three genes was found to be limited to a single cell type during the 5–6 day period from late meiosis to immature pollen formation.     

Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.