Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

Expressed Sequences from Conidial, Mycelial, and Sexual Stages ofNeurospora crassa

In theNeurosporaGenome Project at the University of New Mexico, expressed sequence tags (ESTs) corresponding to three stages of the life cycle of the filamentous fungusNeurospora crassaare being analyzed. The results of a pilot project to identify expressed genes and determine their patterns of expression are presented. 1,865 partial complementary DNA (cDNA) sequences for 1,409 clones were determined using single-pass sequencing. Contig analysis allowed the identification of 838 unique ESTs and 156 ESTs present in multiple cDNA clones. For about 34% of the sequences, highly or moderately significant matches to sequences (of known and unknown function) in the NCBI database were detected. Approximately 56% of the ESTs showed no similarity to previously identified genes. Among genes with assigned function, about 43.3% were involved in metabolism, 32.9% in protein synthesis and 8.4% in RNA synthesis. Fewer were involved in defense (6%), cell signalling (3.4%), cell structure (3.4%) and cell division (2.6%).     

Construction and profiling of a cDNA library from young fruit of satsuma mandarin

To profile gene expression in the early stage of fruit development from ‘Nichinan No. 1’ satsuma mandarin (Citrus unshiu Marc.), we isolated total mRNA at 30 d after flowering. A cDNA library was prepared from mature mRNAs and a total of 2350 cDNA clones were partially sequenced. In all, 1914 ESTs were acquired after the removal of the vector sequence and filtering over a minimum length of 150 nucleotides. A total of 763 unigenes, consisting of 138 contigs and 625 singletons, was identified after assembly of those ESTs. According to our homology search with BLASTX against the NCBI database, the deduced amino acid sequences of 253 unigenes were homologous to proteins with known function and 242 unigenes were significantly matched to proteins with putative or unknown functions. The remaining 268 showed no significant similarity to any protein sequences found in the public database with matches higher than an E value of 10^-5. The 253 unigenes matched to proteins with known function were then manually assigned to 10 cellular functional categories using a modified MIPS MATDB classification. The expression level of each gene was analyzed based on the redundancy of cDNA clones in each contig that comprised more than 10 ESTs. Here, the most abundant gene expressed in young fruits was for a chitinase precursor. A miraculin-like protein and a lectin-related protein precursor were also abundant.     

Analysis of Medicago truncatula nodule expressed sequence tags

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector lambdaHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5' ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.     

Mapping of 22 expressed sequence tags isolated from a porcine small intestine cDNA library

Complementary DNA sequences were selected from a resource of tentatively identified clones from a porcine small intestine cDNA library. Forty PCR primer pairs were designed to amplify 101–309 base pairs of the 3′ untranslated region of the genes. The PCR conditions were optimized by altering both formamide and magnesium concentrations on samples of pig, mouse, and hamster DNA. Twenty primer pairs that, under stringent conditions, were pig-specific and amplified the expected fragments were chosen for regional assignment in a pig/rodent hybrid cell panel. Furthermore, 22 primer pairs were chosen to amplify DNA from the parental animals of the PiGMaP shared reference families in order to detect possible polymorphisms. Primer pairs that generated polymorphisms were used for genetic mapping. A total of 22 porcine expressed sequence tags (ESTs) were cytogenetically or genetically mapped by this approach. Twelve of the mapped ESTs could be added to the human–porcine comparative map. Received: 8 December 1996 / Accepted: 31 January 1997     

Screening and analysis of differentially expressed genes from an alien addition line of wheat Thinopyrum intermedium induced by barley yellow dwarf virus infection.

The alien addition line TAI-27 contains a pair of chromosomes of Thinopyrum intermedium that carry resistance against barley yellow dwarf virus (BYDV). A subtractive library was constructed using the leaves of TAI-27, which were infected by Schizaphis graminum carrying the GAV strain of BYDV, and the control at the three-leaf stage. Nine differentially expressed genes were identified from 100 randomly picked clones and sequenced. Two of the nine clones were highly homologous with known genes. Of the remaining seven cDNA clones, five clones matched with known expressed sequence tag (EST) sequences from wheat and (or) barley whereas the other two clones were unknown. Five of the nine differentially expressed sequences (WTJ9, WTJ11, WTJ15, WTJ19, and WTJ32) were highly homologous (identities >94%) with ESTs from wheat or barley challenged with pathogens. These five sequences and another one (WTJ18) were also highly homologous (identities >86%) with abiotic stress induced ESTs in wheat or barley. Reverse Northern hybridization showed that seven of the nine differentially expressed cDNA sequences hybridized with cDNA of T. intermedium infected by BYDV. Three of these also hybridized with cDNA of line 3B-2 (a parent of TAI-27) infected by BYDV. The alien chromosome in TAI-27 was microdissected. The second round linker adaptor mediated PCR products of the alien chromosomal DNA were labeled with digoxygenin and used as the probe to hybridize with the nine differentially expressed genes. The analysis showed that seven differentially expressed genes were homologous with the alien chromosome of TAI-27. These seven differentially expressed sequences could be used as ESTs of the alien chromosome of TAI-27. This research laid the foundation for screening and cloning of new specific functional genes conferring resistance to BYDV and probably other pathogens.     

Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus

A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with aflatoxin biosynthesis.