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.     

Molecular characterization of an anther-preferential gene from rice

Expression levels of anther-expressed genes in rice were estimated by plaque hybridization. A total of 33 cDNAs, isolated randomly from an anther-enriched cDNA library, were used as probes to hybridize both anther and leaf cDNAs. The expression level of individual cDNA clones was then estimated by counting the number of plaques hybridized to each probe. Based on abundance patterns that appeared in both anther and leaf cDNA libraries, the clones were classified into three groups. This classification showed that the majority of the clones (one group) exhibited expression in both cDNA libraries at almost equal frequency. The other two groups showed either low or no expression in the leaf cDNA library. Among the cDNA clones,RA1003 (detected only in the anther cDNA library) was selected and further characterized at the molecular level. Consistent with the results of the plaque hybridization experiment, northern blot analysis also revealed no gene expression in vegetative organs, leaves, or roots. However, expression was high in the flowers, especially in the anthers. Detailed molecular studies of the gene are also described and discussed here.     

Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs

A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.     

Analysis of cDNA sequences from mouse testis

Few mammalian proteins involved in chromosome structure and function during meiosis have been characterized. As an approach to identify such proteins, cDNA clones expressed in mouse testis were analyzed by sequencing and Northern blotting. Various cDNA library screening methods were used to obtain the clones. First, hybridization with cDNA from testis or brain allowed selection of either negative or differentially expressed plaques. Second, positive plaques were identified by screening with polyclonal antisera to prepubertal testis nuclear proteins. Most clones were selected by negative hybridization to correspond to a low abundance class of mRNAs. A PCR-based solid-phase DNA sequencing protocol was used to rapidly obtain 306 single-pass cDNA sequences totaling more than 104 kb. Comparison with nucleic acid and protein databases showed that 56% of the clones have no significant match to any previously identified sequence. Northern blots indicate that many of these novel clones are testis-enriched in their expression. Further evidence that the screening strategies were appropriate is that a high proportion of the clones which do have a match encode testisenriched or meiosis-specific genes, including the mouse homolog of a rat gene that encodes a synaptonemal complex protein.The nucleotide sequence data reported in this paper have been submitted to Genbank and have been assigned the accession numbers L26606–1.26848.     

47个早期人胚胎低丰度表达基因ESTs筛选及结果分析

构建高质量ｃＤＮＡ文库在基因克隆、ｍＲＮＡ差异展示、表达序列标签测序和基因定位等研究中具有十分重要的作用。为了从早期胚胎中分离人类新基因,构建了受精后３周龄的人ｃＤＮＡ文库,用标记的一链ｃＤＮＡ探针对该文库的６５０８个克隆子进行菌落原位杂交,得到１６７７个无任何杂交信号的低丰度表达克隆子,从中随机挑选了４７个进行５′端部分测序,将测序结果与三大基因库进行序列同源性比较,发现１８个克隆（３８．３％）     

Gene note. An alcohol dehydrogenase-like gene is specifically expressed in potato pistils

The reproductive function of the pistil requires the production of compounds essential for pollen tube growth. A cold-plaque screening of a pollinated pistil cDNA library of Solanum tuberosum resulted in the isolation of cDNA clone cp67. Northern blot analyses revealed that cp67 is specifically expressed in potato pistils and in a limited number of plant species. The deduced CP67 protein displays similarity to long-chain zinc-containing alcohol dehydrogenases (ADHs), although at a similarity level much lower than between other plant ADHs.     

Selective enrichment of cDNAs from salt-stress-induced genes in the wheatgrass, Lophopyrum elongatum, by the formamide-phenol emulsion reassociation technique

We present a novel technique for the enrichment of cDNA libraries to enhance the abundance of clones of differentially expressed genes. The technique is relatively simple, requires moderate quantities of poly(A) + RNA and results in preferential enrichment of clones derived from mRNAs that were of low abundance in their original population. This method was used to isolate cDNA clones of salt-stress-induced genes in the roots of Lophopyrum elongatum, a highly salt-tolerant wheatgrass. An excess of sonicated plasmid DNA from a cDNA library from nonstressed roots was hybridized in a formamide-phenol emulsion with inserts from a cDNA library of stressed roots. Clones that were more abundant in, or were unique to, the library of the stressed roots were recovered as double-stranded fragments by virtue of reconstituted restriction-enzyme-digested ends by ligating them to a plasmid vector. The resulting enriched library was screened by differential colony hybridization and clones of eleven different genes that were more strongly expressed in stressed roots than in controls were selected.     

Identification and molecular characterization of novel anther-specific genes in Oryza sativa L. by using cDNA microarray

The complicated genetic pathway regulates the developmental programs of male reproductive organ, anther tissues. To understand these molecular mechanisms, we performed cDNA microarray analyses and in situ hybridization to monitor gene expression patterns during anther development in rice. Microarray analysis of 4,304 cDNA clones revealed that the hybridization signal of 396 cDNA clones (271 non-redundant groups) increased more than six-fold in every stage of the anthers compared with that of leaves. Cluster analysis with the expression data showed that 259 cDNA clones (156 non redundant groups) were specifically or predominantly expressed in anther tissues and were regulated by developmental stage-specific manners in the anther tissues. These co-regulated genes would be important for development of functional anther tissues. Furthermore, we selected several clones for RNA in situ hybridization analysis. From these analyses, we found several novel genes that show temporal and spatial expression patterns during anther development in addition to anther-specific genes reported so far. These results indicate that the genes identified in this experiment are controlled by different programs and are specialized in their developmental and cell types.     

Vimentin cDNA clones covering the complete intermediate-filament protein are found in an EHS tumor cDNA library

Intermediate filaments are part of the cytoskeleton of most cells. To analyze changes in intermediate filament synthesis, we have isolated two cDNA clones (pV-C25, pV-C877) that cover the complete coding sequence of the murine intermediate filament protein vimentin. The cDNA clones were isolated from a murine Engelbreth-Holm-Swan (EHS) tumor cDNA library by screening under (i) non-stringent conditions with a synthetic oligodeoxynucleotide (oligo), LW-36, which is specific for type-IV collagen, and (ii) stringent conditions with oligo LW75, which was derived from the vimentin clone pV-C25. The cDNA clones contain 38 nucleotides (nt) of the 5'-untranslated region, 1398 nt of the coding region and 7 nt of the 3'-untranslated region. Comparing the mouse sequence with the published sequence for vimentin from hamster, human and chicken, we find shared identities of 99, 97 and 87%, respectively. Since the cDNA clones have been isolated from a basement membrane tumor (EHS) cDNA library, we measured the vimentin mRNA production in EHS tumor cells in culture, and found that this mRNA is half as abundant as mRNA for type-IV mRNA.     

Gene expression in rat dermal papilla cells: analysis of 2529 ESTs

Dermal papilla (DEPA) cells are resident at the base of hair follicles and are fundamental to hair growth and development. Cultured DEPA cells, in contrast to normal fibroblast cells, are capable of inducing de novo hair follicle growth in vivo. By differential screening of a DEPA cDNA library, we have demonstrated that dermal papilla cells are different from fibroblasts at the molecular level. We further studied these cells by random sequencing of 5130 clones from the DEPA cDNA library. Fifty percent had a BLASTX E value < or =1 x 10(-25). Twenty-one percent had similarity to proteins involved in cell structure/motility with 4 of the top 10 most abundant clones encoding extracellular matrix proteins. Clones encoding growth factor molecules were also abundant. The remaining 50.7% of clones had low similarity scores, demonstrating many novel molecules. For example, we identified a new CTGF family member, the rat homologue of Elm1.     

Isolation and characterization of cDNA clones for castration-induced mRNAs in the rat ventral prostate.

To identify gene products involved in castration-induced involution of the rat ventral prostate, we constructed a subtraction cDNA library of the ventral prostate from rats castrated for 48 h. The library was screened with subtracted cDNA probes enriched for sequences with a low copy number expressed in intact or castrated rats. As a result of differential screening, 48 cDNA clones representing 10 different induced mRNAs were isolated. The time course of these mRNA inductions after castration was examined. Within the first 24 h after castration, the level of mRNAs for these cDNA clones was significantly increased and it reached its peak by 48-72 h after castration. Although mRNAs for these cDNA clones were expressed in various tissues from intact rats, an increase in mRNA as a response to castration was observed only in the ventral prostate. Partial sequence analyses of the 10 cDNA clones indicate that three cDNA clones represent rat glutathione S-transferase Yb-1, Yb-2 and Yb-3 subunit mRNA sequences, but for others respective homologues could not be found in a search of the GenBank database (release 67).     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.     

Application for PCR technology to subtractive cDNA cloning: identification of genes expressed specifically in murine plasmacytoma cells.

We describe a simple method for preparing a renewable source of subtractive cDNA which can be used as a hybridization probe or as insert which can be cloned into a variety of convenient vectors. This has been done by ligating a double-stranded oligonucleotide to each end of double-stranded subtractive cDNA, and then using this oligonucleotide sequence to amplify the heterogeneous population of cDNA molecules using the polymerase chain reaction and thermostable Taq DNA polymerase. This method improves the chances for identifying cDNA clones representing low abundance mRNAs that are expressed differentially. Using this approach, we have identified cDNA clones which detect three different low abundance mRNAs that are expressed in mouse plasmacytoma cell lines but not in mouse pre-B or B lymphoma cell lines.     

噬菌体cDNA文库筛选方法的改良

cDNA文库的构建和简便、快速的筛选是获得全长基因的重要途径,基于PCR的筛库方法具有快捷、灵敏的特点。研究改进了基于PCR的噬菌体cDNA文库筛选方法,用液体分装的方法,替代了文库筛选的关键步骤——涂板分区,省去了噬菌体文库铺平板、浸染、培养、划块洗脱的操作过程,使筛库的工作量减少,进一步提高了筛选速度和获得阳性克隆的效率。