A buffalo oocyte-specific subtracted cDNA library was constructed to identify exclusively or preferentially oocyte-expressed genes. The library represented an enriched population of transcripts obtained from oocytes of diverse ovarian follicular origin and at different stages of in vitro maturation. A total of 1173 high-quality sequences of oocyte-specific genes were clustered into 645 unique sequences, out of which 65.76% were represented as singlets and 34.26% as contig expressed sequence tags (ESTs; clusters). Analysis of sequences revealed that 498 of these sequences were identified as a known sequence in mammalian species including buffalo, 103 as uncharacterized ESTs and 44 unknown sequences including 1 novel EST, so far not reported in any species. Gene ontology annotation classified these sequences into functional categories of cellular events and biological processes associated with oocyte competence. Expression status of the isolated unknown ESTs confirmed that many of these are expressed in oocytes exclusively and in others preferentially, some in excess of 80-fold greater in comparison with a variety of somatic tissues. The isolated novel EST was detected to be expressed exclusively in oocytes and testicular cells only. To our knowledge, this is the first report giving a detailed transcriptome account of oocyte-expressed genes in buffalo. This study will provide important information on the physiological control of oocyte development, as well as many questions yet to be addressed on the reproductive process of buffalo.

A cDNA library from white alpaca (Vicugna pacos) skin was constructed using SMART technology to investigate the global gene expression profile in alpaca skin and identify genes associated with physiology of alpaca skin and pigmentation. A total of 5359 high-quality EST (expressed sequence tag) sequences were generated by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 3504 unique sequences including 739 contigs (assembled from 2594 ESTs) and 2765 singletons. BLAST analysis against GenBank nr database resulted in 1287 significant hits (E-value < 10−10), of which 863 were annotated through gene ontology analysis. Transcripts for genes related to fleece quality, growth and coat color (e.g. collagen types I and III, troponin C2 and secreted protein acidic and rich in cysteine) were abundantly present in the library. Other genes, such as keratin family genes known to be involved in melanosome protein production, were also identified in the library. Members (KRT10, 14 and 15) of this gene family are evolutionarily conserved as revealed by a cross-species comparative analysis. This collection of ESTs provides a valuable resource for future research to understand the network of gene expression linked to physiology of alpaca skin and development of pigmentation.

The lower termite, Coptotermes gestroi (Isoptera: Rhinotermitidae), is originally from Southeast Asia and has become a pest in Brazil. The main goal of this study was to survey C. gestroi transcriptome composition. To accomplish this, we sequenced and analyzed 3003 expressed sequence tags (ESTs) isolated from libraries of worker heads. After assembly, 695 uniESTs were obtained from which 349 have similarity with known sequences. Comparison with insect genomes demonstrated similarity, primarily with genes from Apis mellifera (28%), Tribolium castaneum (28%) and Aedes aegypti (10%). Notably, we identified two endogenous cellulases in the sequences, which may be of interest for biotechnological applications. The results presented in this work represent the first genomic study of the Asian subterranean termite, Coptotermes gestroi.

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20 000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.

An immunodominant antigen of Dirofilaria immitis was studied using recombinant DNA techniques. The mRNA from D. immitis adult female worms was translated in vitro and a major 34 k Da antigenic polypeptide product was demonstrated by immunoprecipitation. cDNA was synthesized from mRNA and a λ gt11 expression library was constructed and immunoscreened with dirofilariasis positive serum. A positive clone containing a nearly full length cDNA was isolated. The cDNA was 2415 bp in length and consider of a single open reading frame followed by a long 3' non-coding region of 1446 bp. The open reading frame of 969 bp encoded a polypeptide of 322 amino acids with a molecular weight of 34 400. A cDNA fusion protein synthesized by bacteria (Escherichia coli JM109) using the expression vector pGEMEX-1 was identified as an immunodominant antigen by absorption experiments and had no cross-reactivity with sera from patients with other filarial species.

The construction of two cDNA libraries from young shoots of tea plant [Camellia sinensis cv. Longjing 43 and Anji Baicha], the sequencing of Longjing 43 cDNA clones and the analysis of expressed sequence tags (ESTs) are reported. Totally, 4320 clones from the cDNA library of Longjing 43 were sequenced, and 2963 useful sequences were obtained, corresponding to 68.6% of clones. A total of 416 clones shorter than 150 bp and 863 repeated clones were excluded, the first 1684 valid tea plant ESTs were generated. Most of the ESTs were between 300 and 700 bp, with an average of 478 bp. Six hundred and seven ESTs with known function or putative function were identified by BlastN searches against the National Center of Biotechnology Institute (NCBI) non-redundant nucleotide databases, corresponding to more than 130 functional genes in the tea plant. The rest, 1077 ESTs, were novel gene partial or full sequences. The results indicated that EST sequencing was a rapid and effective approach to identification of novel functional genes for tea plant.

Gene expression in Fucus vesiculosus L. from the Ria Formosa, Faro, Portugal was investigated by screening a cDNA library generated by suppression subtractive hybridization (SSH) for algae undergoing mild desiccation stress (60–70% tissue water content). The subtractive library was constructed from small amounts (1 μg) of total RNA using PCR-based techniques. Screening by reverse Northern analysis (cDNA Southerns) revealed that between 60% and 70% of clones randomly selected from the library were differentially regulated in desiccated and hydrated algae. Most genes could not be directly identified based on sequence homology with known gene sequences. However, several cDNAs for chloroplast-encoded transcripts were identified and shown to be up-regulated or differentially regulated in desiccated algae relative to hydrated controls. These included partial sequences for ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco; rbcL/rbcS), chloroplast coupling factor ATPase (atpH/atpI) and a photosystem I P700 chlorophyll a binding protein (psaA). The usefulness of reverse Northern analysis was confirmed with conventional Northern analysis of transcript abundance for Rubisco, which varied rapidly in response to light and the hydration status of the algae. These results show that SSH is a useful technique for analysing the responses of gene expression to environmental stress, and as a starting point for the subsequent identification of stress-responsive genes in macroalgae.