Plasmodium falciparum: generation of a cDNA library enriched in sporozoite-specific transcripts by directional tag subtractive hybridization

We have adapted the "directional tag subtractive hybridization" technique as a means of investigating stage-specific gene expression in Plasmodium falciparum. This technique utilizes unidirectional cDNA libraries cloned into separate lambda vectors and involves hydroxyapatite chromatographic separation of target antisense cDNA and driver sense strand cRNA followed by PCR amplification of cDNA sequences specific to the target stage. This technique enabled efficient subtraction of asexual blood stage sequences from a P. falciparum sporozoite cDNA library and led to identification of novel sporozoite sequences. This technique can be applied to study gene expression in parasite stages that are difficult to obtain routinely.     

Prokaryotic Suppression Subtractive Hybridization PCR cDNA Subtraction, a Targeted Method To Identify Differentially Expressed Genes

Molecular biology tools can be used to monitor and optimize biological treatment systems, but the application of nucleic acid-based tools has been hindered by the lack of available sequences for environmentally relevant biodegradation genes. The objective of our work was to extend an existing molecular method for eukaryotes to prokaryotes, allowing us to rapidly identify differentially expressed genes for subsequent sequencing. Suppression subtractive hybridization (SSH) PCR cDNA subtraction is a technique that can be used to identify genes that are expressed under specific conditions (e.g., growth on a given pollutant). While excellent methods for eukaryotic SSH PCR cDNA subtraction are available, to our knowledge, no methods previously existed for prokaryotes. This work describes our methodology for prokaryotic SSH PCR cDNA subtraction, which we validated using a model system: Pseudomonas putida mt-2 degrading toluene. cDNA from P. putida mt-2 grown on toluene (model pollutant) or acetate (control substrate) was subjected to our prokaryotic SSH PCR cDNA subtraction protocol to generate subtraction clone libraries. Over 90% of the sequenced clones contained gene fragments encoding toluene-related enzymes, and 20 distinct toluene-related genes from three key operons were sequenced. Based on these results, prokaryotic SSH PCR cDNA subtraction shows promise as a targeted method for gene identification.     

Suppressive Subtraction Libraries to Identify Interferon-Inducible Genes in Fish

Previous attempts to identify genes in fish that respond to virus infection or interferon induction have not been particularly productive. Since these genes are very important in developing strategies to control disease outbreaks in aquaculture, we began a study of interferon-inducible genes in fish using suppressive subtraction hybridization to construct cDNA libraries enriched for interferon-inducible genes. Subtraction hybridization libraries were constructed with cDNA obtained from the kidney, spleen, and liver of Chinook salmon (Oncorhynchus tshawytscha) and staghorn sculpin (Hemilepidotus spinosus) before and after injection with poly IC, a potent interferon inducer. The ``identified' genes in both cDNA libraries corresponded to previously identified genes of the fish complement system, the interferon-inducible proteins observed in mammalian cells, and the Vig-1 gene, identified in fish cells after infection with fish rhabdoviruses. Received June 19, 2001; accepted July 13, 2001     

A subtractive hybridisation method for the enrichment of moderately induced sequences.

Moderately induced genes often escape detection in conventional subtraction hybridisation cloning. Here a modification of a phagemid subtraction protocol is described that overcomes this problem. The protocol uses low ratio hybridisation of driver to target sequences to allow enrichment of the sequences of interest, and back-hybridisation of the subtracted sequences with induced sequences to reduce the accumulation of false positive clones. The procedure takes advantage of the quantitative representation of cellular RNA populations in cDNA libraries, therefore, they may serve not only as renewable sources of driver and target sequences, but also as sources of population cRNAs used in northern blots and differential Southern blots.     

Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions

The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferoxamine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.     

Gene expression profiles linked to the hormonal induction of male-effect pheromone synthesis in goats (Capra hircus)

The male effect is a well-known phenomenon in female sheep and goats whereby a pheromone-induced activation of reproductive function occurs. However, the molecule(s) involved in this phenomenon are unknown. We investigated gene expression profiles for the induction of male effect pheromone synthesis using a PCR-based cDNA subtraction strategy. We constructed two subtracted cDNA libraries using mRNA from the skin of the head or rump region of orchidectomized male goats with or without pheromone induction using testosterone or dihydrotestosterone (DHT). Both libraries were assumed to contain genes whose expression increases with pheromone induction. Clones (n = 480) from each library were sequenced and identified using BLAST to reveal 115 and 239 types of sequences in the libraries of the head and rump region, respectively. Among these, 12 genes were expressed in both libraries. We conducted real-time PCR to further analyze their expression using cDNA samples derived from pheromone-producing or nonproducing skin from the head of an ovariectomized female goat with or without DHT implantation, respectively. For nine genes, we observed significantly increased expression in samples following DHT implantation. Among these, stearoyl-CoA desaturase 1 (SCD1) and elongation of long chain fatty acids family member 5 (ELOVL5) genes showed more than 100-fold higher expression levels in pheromone-positive samples, suggesting that the products of these genes may be important in pheromone synthesis.     

cDNA representational difference analysis of ileal Peyer's patches in lambs after oral inoculation with scrapie

cDNA representational difference analysis (RDA) was used to study gene expression profiles in the ileal Peyer's patch of a lamb 1 week after oral inoculation with the scrapie agent. Twenty-five differentially expressed cDNA fragments were identified and cloned. Sequence analysis indicated seven novel gene sequences. Other clones shared sequence homology with genes encoding ribosomal and mitochondrial proteins, the translation initiation factor EIF4GII and the bovine pancreatic thread protein. Reverse Northern was used to confirm the differential expression in another four lambs inoculated with scrapie and the tissue distribution of the novel genes was examined using Northern blot analysis.     

Applying genomics to the avian inner ear: development of subtractive cDNA resources for exploring sensory function and hair cell regeneration

We applied a micro-cDNA-based subtraction method to identify genes expressed in the regenerating sensory epithelia (SE) of the chicken inner ear. Sensory hair cells in the avian utricle SE are in a constant state of turnover, where dying hair cells are replaced by new ones derived from supporting cells. In contrast, hair cells in the cochlea remain quiescent unless damaged. We used this difference to enrich for utricle-specific genes, using reiterative cDNA subtraction and demonstrate enrichment for utricle-specific sequences. A total of 1710 cDNA sequence reads revealed the presence of many cDNAs encoding known structural components of the SE (for example, Harmonin and beta-tectorin), proteins involved in cellular proliferation, such as P311, HIPK2, and SPALT1, among many others of unknown function. These libraries are the first of their kind and should prove useful for the discovery of candidate genes for hearing disorders, regenerative and apoptotic pathways, and novel chicken ESTs.     

Selection of proteins with desired properties from natural proteome libraries using mRNA display

mRNA display is a powerful yet challenging in vitro selection technique that can be used to identify proteins with desired properties from both natural proteome and combinatorial polypeptide libraries. The physical conjugation between a protein and its own RNA presents unique challenges in manipulating the displayed proteins at a low nanomolar scale in an RNase-free environment. The following protocol outlines the generation of cDNA libraries derived from natural organisms as well as the steps required for generation of mRNA-protein fusion molecules, in vitro functional selection and regeneration of the selected cDNA library. The selection procedures for the identification of protease substrates and Ca(2+)-dependent calmodulin-binding proteins from natural cDNA libraries are presented as examples. The method can be generally applied to the identification of protein sequences with desired properties from various natural proteome libraries. One round of mRNA display-based selection can be accomplished in ~7 d.     

溶藻弧菌诱导凡纳滨对虾消减文库的构建及其表达序列标签分析

利用抑制消减杂交技术构建了溶藻弧菌(Vibrio alginolyticus)诱导的凡纳滨对虾(Litopenaeus vannamei)血淋巴细胞cDNA文库。用DNAMAN5.2.2软件对560条高质量的ESTs进行聚类,共获得239个Unigenes。与GenBank进行BLASTx和BLASTn同源比较,其中66.9%为已知功能基因,33.1%为未知功能基因,GO分类将其分为7类,包括能量和基础代谢类相关的基因为第一大类占36%,免疫相关基因占15%,其他基因占8%,信号转导类占3%,抗氧化酶和凋亡相关蛋白均为2%,核蛋白类占1%。实验结果表明凡纳滨对虾在溶藻弧菌诱导下可产生一系列特异基因的表达,通过对文库的分析显示,基于PCR方法建立的SSH文库为取得大量免疫相关基因的ESTs序列提供了可能。       

Evidence of mitochondrial involvement in scrapie infection.

Two cDNA libraries were constructed from brain membrane and cytoskeletal preparations purified from scrapie-infected hamster brains. Four recombinants strongly preferential to the scrapie cytoskeletal preparation were identified by the differential hybridization of 7,000 recombinants. These clones were not, however, preferential to total nucleic acids extracted from scrapie-infected hamster brains. DNA sequence analysis revealed all four clones to have significant sequence similarities to the mouse mitochondrial genome. This correlation led us to consider a mitochondrial association with scrapie infectivity. Brain mitochondria were purified by sucrose gradient density centrifugation and found to contain high infectivity. Removal of mitochondrial outer membranes by osmotic shock or digitonin treatment resulted in no detectable loss of titer.     

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.     

A family of lambda phage cDNA cloning vectors, lambda SWAJ, allowing the amplification of RNA sequences

This paper describes the construction and characterization of a family of lambda phage cDNA cloning vectors that allows high-efficiency directional cDNA cloning and selective amplification of either sense or antisense cRNA sequences. These vectors contain several unique restriction sites (EcoRI, XbaI, and SacI) positioned between two specific phage promoters, SP6 and T7. This system facilitates the in vitro preparation of single-stranded (ss) RNA molecules that should be useful in subtractive hybridization and in situ hybridization procedures. Using subtractive hybridization and this vector system, it should be possible to identify sequences present in one cDNA library and not another. In addition, it should be possible to construct subtracted cDNA libraries in these vectors and to generate high specific activity, ss, antisense cRNA probes directly from DNA prepared from the whole subtracted library or from individual clones.