Radiation hybrid map assignments of 11 ESTs obtained from a 28-day-old swine embryo cDNA library to the IMpRH map

In order to improve the map resolution and to locate more genes on the porcine radiation hybrid map, expressed sequence tags (ESTs) were isolated from a 28-day-old normal pig embryo cDNA library. The ESTs were sequenced from the 5'-end and similarities were checked with sequences registered in the NCBI DNA database (http://www.ncbi.nlm.nih.gov/blast/). The ESTs sequences which have high identity scores (>80%) against human genes or ESTs were further sequenced from the 3' untranslated region. The ESTs which were sequenced successfully were used to design primers for PCR analysis of the radiation hybrid panel. Eleven ESTs were physically mapped to porcine chromosomes 2, 4, 8, 10, 13, 14 and X. The localizations are in agreement with the comparative mapping data between human and pig. The results will provide unique information to the comparative map of human and pig.     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

Improving the comparative map of SSC2p-q13 by sample sequencing of BAC clones

To improve the comparative map for pig chromosome 2 and increase the gene density on this chromosome, a porcine bacterial artificial chromosome (BAC) library was screened with 17 microsatellite markers and 18 genes previously assigned to pig chromosome 2. Fifty-one BAC clones located in the region of a maternally imprinted quantitative trait locus for backfat thickness (BFT) were identified. From these BACs 372 kb were sample sequenced. The average read length of a subclone was 442 basepair (bp). Contig assembly analysis showed that every bp was sequenced 1.28 times. Subsequently, sequences were compared with sequences in the nucleotide databases to identify homology with other mammalian sequences. Sequence identity was observed with sequences derived from 35 BACs. The average percentage identity with human sequences was 87.6%, with an average length of 143 bp. In total, sample sequencing of all BACs resulted in sequence identity with 29 human genes, 13 human expressed sequence tags (ESTs), 17 human genomic clones, one rat gene, one porcine gene and nine porcine ESTs. Eighteen genes located on human chromosome 11 and 19, and seven genes from other human locations, one rat gene and one porcine gene were assigned to pig chromosome 2 for the first time. The new genes were added to the radiation hybrid map at the same position as the locus from which the BAC that was sequenced was derived. In total 57 genes were placed on the radiation hybrid map of SSC2p-q13.     

Large-scale sequencing of normalized full-length cDNA library of soybean seed at different developmental stages and analysis of the gene expression profiles based on ESTs

Although GenBank has now covered over 1,400,000 expressed sequence tags (ESTs) from soybean, most ESTs available to the public have been derived from tissues or environmental conditions rather than developing seeds. It is absolutely necessary for annotating the molecular mechanisms of soybean seed development to analyze completely the gene expression profiles of its immature seed at various stages. Here we have constructed a full-length-enriched cDNA library comprised of a total of 45,408 cDNA clones which cover various stages of soybean seed development. Furthermore, we have sequenced from 5′ ends of these clones, 36,656 ESTs were obtained in the present study. These EST sequences could be categorized into 27,982 unigenes, including 22,867 contigs and 5,115 singletons, among which 27,931 could be mapped onto soybean 20 chromosome sequences. Comparative genomic analysis with other plants has revealed that these unigenes include lots of candidate genes specific to dicot, legume and soybean. Approximately 1,789 of these unigenes currently show no homology to known soybean sequences, suggesting that many represent mRNAs specifically expressed in seeds. Novel abundant genes involved in the oil synthesis have been found in this study, may serve as a valuable resource for soybean seed improvement.     

The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.     

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.Communicated by R. Hagemann     

Mapping of genes expressed in activated porcine Peyer's patch

To determine the chromosomal locations for genes expressed in porcine Peyer's patches, polymerase chain reaction-based mapping of expressed sequence tags (ESTs) isolated from a porcine Peyer's patch-specific cDNA library was performed across a 6500-rad swine radiation hybrid panel. A total of 116 ESTs were mapped with LOD scores >6.0, and another 11 ESTs had LOD scores between 5.0 and 6.0. Of these 127 ESTs, 63% matched known genes (相似文献   

Genes expressed in sugarcane maturing internodal tissue

To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.     

Comparative mapping in the pig: localization of 214 expressed sequence tags

In total, 214 ESTs (Expressed Sequence Tags) were assigned to the porcine gene map by using somatic cell hybrid mapping, radiation hybrid mapping, and FISH. The ESTs were isolated from a porcine small intestine cDNA library on the basis of significant sequence identity with human annotated genes. In total, 390 primer pairs were designed primarily in the 3' UTR of the sequences. Overall, 58.6% of the ESTs were successfully mapped by this approach. In total, 191 of the localizations are in agreement with the human comparative map, strongly indicating that these represent true orthologous genes. The remaining 23 ESTs provide new comparative mapping data, which should be considered as preliminary until confirmed by other studies. Our mapping efforts provide a significant contribution to the porcine map as well as to the comparative map for human and pig.     

Generation of 919 expressed sequence tags from immature flower buds and gene expression analysis using expressed sequence tags in the model plant Lotus japonicus

Lotus japonicus has received increased attention as a potential model legume plant. In order to study gene expression in reproductive organs and to identify genes that play a crucial function in sexual reproduction, we constructed a cDNA library from immature flower buds containing anthers at the stage of developing tapetum cells in L. japonicus, and characterized 919 expressed sequence tags (ESTs) randomly selected from a cDNA library of the immature flower buds. The 919 ESTs analyzed were clustered into 821 non-redundant EST groups. As a result of a database search, 436 groups (53%) out of the 821 groups showed sequence similarity to genes registered in the public database. Out of these 436 groups, 109 groups showed similarity to genes encoding hypothetical proteins whose function had not yet been estimated. Three hundred eighty five groups (47%) showed no significant homology to known sequences and were classified as novel sequences. A comparison of 821 non-redundant EST sequences and EST sequences derived from the whole plant L. japonicus revealed that 474 EST sequences derived from immature flower buds were not found in the EST sequences of the whole plant. In order to confirm the expression pattern of potential reproductive-organ specific EST clones, nine clones, which were not matched to ESTs derived from the whole plant, were selected, and RT-PCR analysis was performed on these clones. As a result of RT-PCR, we found two novel anther specific clones. One clone was homologous to a gene encoding human cleft lip and palate associated transmembrane protein (CLPTM1) like protein, and the other clone did not show a significant similarity to any genes deposited in the public database. These results indicate that ESTs analyzed here represent a valuable resource for finding reproductive-organ specific genes in Lotus japonicus.     

An EST survey of the sugarcane transcriptome

Its large genome and high polyploidy makes sugarcane (Saccharum spp.) a singularly challenging crop to study and improve using genetic approaches. To provide large numbers of functionally characterized candidate genes that might be tested for direct association (rather than distant linkage) with economically important traits, we sequenced the 5' ends of 9,216 clones from three cDNA libraries (apex, leaf and mature internode), representing 3,401 non-redundant sequences. About 57% of these sequences could be assigned a tentative function based on statistically significant similarity to previously characterized proteins or DNA sequences. Another 28% corresponded to previously identified, but uncharacterized, sequences. Some of the remaining unidentified sequences were predicted to be genes which could potentially be new to plants or unique to sugarcane. Comparisons of the sugarcane ESTs to a large sorghum EST database revealed similar compositions of expressed genes between some different tissues. Comparison to a detailed Arabidopsis protein database showed some highly conserved sequences, which might be useful DNA markers for pan-angiosperm comparative mapping. These EST sequences provide a foundation for many new studies to accelerate isolation of agronomically important genes from the cumbersome sugarcane genome.     

Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 13,000 cDNA clones from two time-of-day-specific libraries (morning and evening library) generating approximately 20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For approximately 50% of the sequences (710 of 1431), significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function) were detected. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST sequences. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their expression patterns over the course of the day. Nevertheless, we were able to identify four new clock-controlled genes. On the basis of these data we predict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.     

兔单个植入前克隆胚胎cDNA文库的构建

人与小鼠和牛在正常胚胎植入前发育过程中基因活化的研究已经取得了长足的进展 ,但是还没有对同期克隆胚胎相关研究的报道 .利用单个家兔植入前移核重构胚胎成功地构建了MⅡ卵母细胞及发育至 4 、8 细胞期的胚胎和囊胚的特异性cDNA文库 .并用 β肌动蛋白和LAPTM4α证实这类文库是可靠的 .以 8 细胞期移核重构胚cDNA文库为例 ,随机挑取克隆进行测序分析 :其中 2 3的基因EST片段可以在GenBank或EST库中找到同源序列 ,约 1 3的EST片段属于未知的新片段 ,表明这是一类重要的新兴基因资源库 (期特异性EST库 ) .这种利用单胚胎构建cDNA文库的方法 ,解决了胚胎研究材料受限的问题 ,在时间上更加精确 ,更符合胚胎发育的规律 ,也能够更加准确地反映出一些克隆胚胎的异常表型 ,是研究早期胚胎发育基因表达以及克隆胚胎再程序化基因表达的一种有效手段 .