Differential expression of house-keeping genes of Aspergillus nidulans during sexual development

The rpl3 gene and the rpl37 gene for Aspergillus nidulans ribosomal protein L3 (RPL3) and RPL37, which were identified as located on chromosome I and chromosome III, respectively, were isolated from chromosome-specific cosmid libraries. The nucleotide sequences of both of the rpl3 gene and the rpl37 gene identified the ORFs of 392 amino acids and 92 amino acids, respectively. Both of the two genes were present in a single copy. The expression of both genes together with two other house-keeping genes, the rps16 gene for RPS16 and the gene for gamma-actin, was analyzed during sexual development. All four genes showed nearly identical expression patterns in that each gene expression reached its maximum after 2 h, decreased thereafter, and increased again after 30-40 h of induction of sexual development.     

Cloning and characterization of chitin synthase gene fragments from Penicillium chrysogenum

Abstract We constructed a 3'-directed cDNA library of cleistothecia and Hülle cells of Aspergillus nidulans to examine gene expression patterns of the sexual structures and to have probes necessary to isolate sexual structure-specific genes. Sequencing of 360 randomly selected cDNA clones yielded 272 expressed sequence tags (ESTs), most of which probably represent frequently or less expressed genes in sexual structures of A. nidulans . Among the 272 ESTs, 33 ESTs (87 cDNA clones) appeared more than once and 2 ESTs appeared 6 times; 9 ESTs matched GenBank entries. When compared with sequences obtained from a mycelial 3'-directed cDNA library of A. nidulans , 28 out of 33 ESTs seem to be sexual structure-specific. Northern blot analyses of 20 ESTs showed that 17 are sexual structure-specific. The remaining three ESTs also hybridized with RNA isolated from vegetative mycelia. These results suggest that analyses of ESTs from different cell types or tissues can readily demonstrate gene expression patterns of specific cell types and identify cell type-specific cDNA probes.     

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.     

Isolation of genes expressed during the developmental stages of the oyster mushroom, Pleurotus ostreatus, using expressed sequence tags

In order to isolate and identify the developmentally regulated genes during fruiting body development, cDNA libraries were constructed from eight developmental stages of the Oyster mushroom, Pleurotus ostreatus. From these libraries, 11 761 expressed sequence tags (PoESTs) were generated. Of these, 4060 different genes (PoUnigenes) were identified, representing 34.5% of the entire genome. Redundancy analysis of ESTs during the developmental stages identified eight, 13 and two genes that were specifically expressed in mycelia, fruiting body and basidiospore, respectively. RT-PCR was used to confirm the specific expression of nine genes which showed specific redundancy in fruiting body stages, four genes of which were expressed specifically in fruiting body stages as expected in redundancy analysis, and other genes were expressed abundantly in fruiting body stages. The EST database of P. ostreatus generated during this study provides a genetic and biochemical basis for future studies of the developmental stages of basidiomycetes.     

Correlated clustering and virtual display of gene expression patterns in the wheat life cycle by large-scale statistical analyses of expressed sequence tags

Compared to rice, wheat exhibits characteristic growth habits and contains complex genome constituents. To assess global changes in gene expression patterns in the wheat life cycle, we conducted large-scale analysis of expressed sequence tags (ESTs) in common wheat. Ten wheat tissues were used to construct cDNA libraries: crown and root from 14-day-old seedlings; spikelet from early and late flowering stages; spike at the booting stage, heading date and flowering date; pistil at the heading date; and seeds at 10 and 30 days post-anthesis. Several thousand colonies were randomly selected from each of these 10 cDNA libraries and sequenced from both 5' and 3' ends. Consequently, a total of 116 232 sequences were accumulated and classified into 25 971 contigs based on sequence homology. By computing abundantly expressed ESTs, correlated expression patterns of genes across the tissues were identified. Furthermore, relationships of gene expression profiles among the 10 wheat tissues were inferred from global gene expression patterns. Genes with similar functions were grouped with one another by clustering gene expression profiles. This technique might enable estimation of the functions of anonymous genes. Multidimensional analysis of EST data that is analogous to the microarray experiments may offer new approaches to functional genomics of plants.     

Characterization of Solt, a novel SoxLZ/Sox6 binding protein expressed in adult mouse testis

SoxLZ/Sox6, a member of the Sox protein family, contains a leucine zipper motif in addition to an HMG box, which is its DNA binding domain. Here we have identified a novel SoxLZ/Sox6 binding protein, termed Solt, which we obtained independently using both a far-Western blot and a yeast two-hybrid screen. Like SoxLZ/Sox6 mRNA, Solt mRNA was exclusively expressed in the testis in mouse. Solt contains an unusual leucine zipper, which bound to the leucine zipper region of SoxLZ/Sox6 in vitro. In transient transfection assays in CHO cells with SoxLZ/Sox6 containing the transactivational region of herpes simplex virus VP16, expression of a reporter gene that carries a cis binding region for Sox proteins was significantly enhanced by the co-expression of Solt and Ca(2+)/calmodulin-dependent protein kinase IV.     

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.     

转化的大鼠胚胎成纤维细胞系差异表达基因的筛选研究

来源于转化的大鼠胚胎成纤维细胞系的两株细胞,A1－5细胞与B4细胞相比表现出非常强的抗辐射性并伴随不同寻常强的G2延迟效应;用PCR选择性抑制消减杂交方法对这两株细胞进行差减,希望找到对A1－5细胞表现出的不同寻常的表型起关键作用的某一个或某一些基因。结果得到了160个差减转化子,逐个进行序列测定,并进行Dot blot杂交,共得到35个差异表达基因片段（EST）。通过对美国国家生物技术信息中心（NCBI）的非冗余序列库（NT）、鼠EST库及人EST库的BLAST进行同源检索,发现其中21个代表了尚未登录的新基因,另外14个分别与已知基因高度同源。     

Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues.

A four-step procedure for the efficient and systematic mining of whole EST libraries for differentially expressed genes is presented. After eliminating redundant entries from the EST library under investigation (step 1), contigs of maximal length are built upon each remaining EST using about 4 000 000 public and proprietary ESTs (step 2). These putative genes are compared against a database comprising ESTs from 16 different tissues (both normal and tumour affected) to determine whether or not they are differentially expressed (step 3; electronic northern). Fisher's exact test is used to assess the significance of differential expression. In step 4, an attempt is made to characterise the contigs obtained in the assembly through database comparison. A case study of the CGAP library NCI_CGAP_Br1.1, a library made from three (well, moderately, and poorly differentiated) invasive ductal breast tumours (2126 ESTs in total) was carried out. Of the maximal contigs, 139 were found to be significantly (alpha = 0.05) over-expressed in breast tumour tissue, while 13 appeared to be down-regulated.     

Identification of Gasz,an evolutionarily conserved gene expressed exclusively in germ cells and encoding a protein with four ankyrin repeats,a sterile-alpha motif,and a basic leucine zipper

To discover causes of infertility and potential contraceptive targets, we used in silico subtraction and genomic database mining to identify conserved genes with germ cell-specific expression. In silico subtraction identified an expressed sequence tag (EST) present exclusively in a newborn mouse ovary library. The full-length cDNA sequence corresponding to this EST encodes a novel protein containing four ankyrin (ANK) repeats, a sterile-alpha motif (SAM), and a putative basic leucine zipper (bZIP) domain. Northern blot and semiquantitative RT-PCR analyses demonstrated that the mRNA is exclusively expressed in the mouse testis and ovary. The expression sites were localized by in situ hybridization to pachytene spermatocytes in the testis and oocytes in the ovary. Immunohistochemistry showed that the novel protein is localized to the cytoplasm in pachytene spermatocytes and early spermatids, oocytes at all stages of oogenesis, and in early preimplantation embryos. Based on its germ cell-specific expression and the presence of ANK, SAM, and basic leucine zipper domains, we have termed this novel protein GASZ. The mouse Gasz gene, which consists of 13 exons and spans 60 kb, is located on chromosome 6 between the Wnt2 and cystic fibrosis transmembrane conductance regulator (Cftr) genes. Using genomic database mining, orthologous genes encoding GASZ were identified in the rat, cow, baboon, chimpanzee, and human. Phylogenetic analyses reveal that the GASZ proteins are highly conserved among these species. Human and mouse GASZ proteins share 85.3% amino acid identity, and human and chimpanzee GASZ proteins differ by only 3 out of 475 amino acids. In humans, the GASZ gene resides on chromosome 7 and is similarly composed of 13 exons. Because both ANK repeats and the SAM domain function as protein-protein interaction modules that mediate signal transduction cascades in some systems, GASZ may represent an important cytoplasmic signal transducer that mediates protein-protein interactions during germ cell maturation in both males and females and during preimplantation embryogenesis.     

Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis

Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.     

Identification of key genes involved in root development of tomato using expressed sequence tag analysis

Root system of plants are actually fascinating structures, not only critical for plant development, but also important for storage and conduction. Due to its agronomic importance, identification of genes involved in root development has been a subject of intense study. Tomato is the one of the most consumed vegetables in the world. Tomato has been used as model system for dicot plants because of its small genome, well-established transformation techniques and well-constructed physical map. The present study is targeted to identify of root specific genes expressed temporally and also gene(s) involved in lateral root and profuse root development. A total of 890 ESTs were identified from five EST libraries constructed using SSH approach which included temporal gene regulation (early and late) and genes involved in morphogenetic traits (lateral and profuse rooting). One hundred sixty-one unique ESTs identified from various libraries were categorized based on their putative functions and deposited in NCBI-dbEST database. In addition, 36 ESTs were selected for validation of their expression by RT-PCR. The present findings will help in shedding light to the unexplored developmental process of root growth in tomato and plant in general.     

Use of a large-scale Triticeae expressed sequence tag resource to reveal gene expression profiles in hexaploid wheat (Triticum aestivum L.).

The US Wheat Genome Project, funded by the National Science Foundation, developed the first large public Triticeae expressed sequence tag (EST) resource. Altogether, 116,272 ESTs were produced, comprising 100,674 5' ESTs and 15 598 3' ESTs. These ESTs were derived from 42 cDNA libraries, which were created from hexaploid bread wheat (Triticum aestivum L.) and its close relatives, including diploid wheat (T. monococcum L. and Aegilops speltoides L.), tetraploid wheat (T. turgidum L.), and rye (Secale cereale L.), using tissues collected from various stages of plant growth and development and under diverse regimes of abiotic and biotic stress treatments. ESTs were assembled into 18,876 contigs and 23,034 singletons, or 41,910 wheat unigenes. Over 90% of the contigs contained fewer than 10 EST members, implying that the ESTs represented a diverse selection of genes and that genes expressed at low and moderate to high levels were well sampled. Statistical methods were used to study the correlation of gene expression patterns, based on the ESTs clustered in the 1536 contigs that contained at least 10 5' EST members and thus representing the most abundant genes expressed in wheat. Analysis further identified genes in wheat that were significantly upregulated (p < 0.05) in tissues under various abiotic stresses when compared with control tissues. Though the function annotation cannot be assigned for many of these genes, it is likely that they play a role associated with the stress response. This study predicted the possible functionality for 4% of total wheat unigenes, which leaves the remaining 96% with their functional roles and expression patterns largely unknown. Nonetheless, the EST data generated in this project provide a diverse and rich source for gene discovery in wheat.