An improved method for subtractive cloning of differentially expressed genes in higher plants by protective exonuclease digestion and discriminating PCR amplification

An improved method for subtractive cloning with enhanced efficiency was developed by modifying the enzymatic degrading subtraction. The thionucleotide-modified tester cDNA fragments under control of one linker-primer were hybridized with excess driver cDNA fragments flanked by the other distinct linker-primer. After selective digestion of incompletely protected tester/driver and of unprotected driver/driver molecules with exonuclease III and VII, the protected tester/tester reassociates due to thionucleotides were exclusively amplified by PCR with the tester-cDNA-specific primer. The subtractively enriched target cDNA fragments, showing distinct bands in an agarose gel, were inserted into pUC19, and random colonies with inserts were screened by Northern hybridization to tester and driver RNA. Four distinct clones were confirmed to be up-regulated by the withdrawal of potassium from the nutrient solution of seedling barley growing hydroponically. The original protocol generated only smeared amplicons due to non-selective PCR amplification of the hybridized cDNA mixture including remains of undigested driver cDNA.Abbreviations EDS Enzymatic degrading subtraction - SET Subtractively enriched target     

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato (Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.     

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.     

A Novel mRNA Level Subtraction Method for Quick Identification of Target-Orientated Uniquely Expressed Genes Between Peanut Immature Pod and Leaf

Subtraction technique has been broadly applied for target gene discovery. However, most current protocols apply relative differential subtraction and result in great amount clone mixtures of unique and differentially expressed genes. This makes it more difficult to identify unique or target-orientated expressed genes. In this study, we developed a novel method for subtraction at mRNA level by integrating magnetic particle technology into driver preparation and tester–driver hybridization to facilitate uniquely expressed gene discovery between peanut immature pod and leaf through a single round subtraction. The resulting target clones were further validated through polymerase chain reaction screening using peanut immature pod and leaf cDNA libraries as templates. This study has resulted in identifying several genes expressed uniquely in immature peanut pod. These target genes can be used for future peanut functional genome and genetic engineering research.     

Multiplicity and diversity of cloned zein cDNA sequences and their chromosomal localisation

We have constructed and screened cDNA libraries from total maize endosperm poly(A) RNA or from a mRNA fraction enriched in zein sequences. From these libraries we have isolated clones representative of the major classes of zein cDNA sequences and have characterised them by crosshybridisation, by hybrid-selected translation, by in situ hybridisation to maize chromosomes, and hybridisation to genomic Southern blots. We conclude that at least four types of non cross-hybridising zein sequences are present, two coding for light chains and two for heavy chains. At least in the case of the light zeins, there is considerable sequence diversity among the clones which hybridise to each type. Similar results are obtained by translation of the mRNAs selected by each clone. In situ hybridisation shows that the light chain zein genes are located on chromosomes 4, 7, and 10, whilst genes coding for some of the heavy chain zeins are confined to the distal part of the long arm of chromosome 4.     

基因组消减杂交技术及其应用

基因组消减杂交是通过突变型（驱赶DNA,driver)与野生型(检测DNA,tester)基因组DNA之间的差异比较来分离和鉴定突变基因的方法,该方法具有简便、快速、敏感、经济等特点,已被广泛应用于分离和鉴定肿瘤缺失和重排基因,制备多态位点探针,分离遗传性疾病、病毒感染性疾病的致病基因和基因诊断等领域．文章对这一新技术的建立和发展、实验策略、应用实例、目前存在的问题和发展前景等方面做了简要的综述．     

cDNA cloning of a human androgen-induced mRNA exhibiting an early and protein synthesis-independent induction

The detailed mechanism of action of androgens remains unknown. We have used an androgen-dependent human prostate cancer cell line and a subtractive cDNA hybridisation strategy to enrich for androgen-dependent sequences. This yielded a cDNA clone which exhibits the characteristics of a primary trans-activated target for androgens. This androgen-regulated gene encodes a polyadenylated 4.5 kb mRNA which is induced 30-50-fold within 3 h of treatment with 10 nM dihydrotestosterone. The induction does not require continued protein synthesis as it is maintained in the presence of protein synthesis inhibitors.     

Identifying the genome of wood barley Hordelymus europaeus (Poaceae: Triticeae)

Wood barley, Hordelymus europaeus, was compared with other Triticeae species by Southern and fluorescence in situ hybridisation using total genomic DNA and repetitive sequences as probes. On Southern blots, the total genomic probe from H. europaeus hybridised strongly to DNA of its own species and to Leymus and Psathyrostachys, indicating the presence of Ns genome in H. europaeus. Furthermore, the total genomic probe from P. fragilis hybridised to DNA of H. europaeus as much as to all of the Psathyrostachys and Leymus species examined. Ns genome-specific DNA sequences isolated from L. mollis (pLmIs1, pLmIs44 and pLmIs53) hybridised essentially to H. europaeus and all of the species of Leymus and Psathyrostachys. Chromosomal localization of these clones on H. europaeus confirmed the presence of Ns genome-specific DNA on all chromosomes, indiscriminately. Under moderate hybridisation stringency the Ns genome-specific probes, together with repetitive sequences pTa71 and pAesKB7, produced species-specific RFLP banding profiles on Southern blots. A phenetic tree based on these profiles revealed a distinct Ns species cluster within the Triticeae, represented by Leymus and Psathyrostachys species. Hordelymus europaeus belonged to this Ns cluster. Chromosomal mapping of the 18S-25S and the 5S ribosomal genes, together with the repetitive sequence pLrTaiI, corroborated that H. europaeus was most probably related to Leymus, especially the European/Eurasian members of sect. Leymus. In an attempt to identify the genome of H. europaeus, different approaches were employed; the results clearly showed that wood barley had the Ns basic genome and nothing else.     

抑制差减杂交法分离玉米幼苗淹水诱导表达基因

以淹水处理（submergence-treated,ST)的玉米（Zea maysL.)幼苗根部cDNA为目标群体,未处理（untreated,UT)的玉米幼苗cDNA为对照群体,进行抑制差减杂交。用经过UT差减的STcDNA构建了一个含有大约2000个独立克隆的差减文库。对随机挑取的408个克隆进行差异筛选。获得了184个在ST中特异表达或表达增强的候选克隆。对其中155个cDNA克隆测序并去除重复克隆后,共得到95个差异表达的cDNA片段。GenBank中BLAST查询结果表明;6个克隆为已知的玉米核苷酸序列;68个克隆与已知基因或EST序列部分区域的同源性为60%-90%;21个克隆在GenBank中无法查到对应的同源序列。可能代表了新基因。或者由于序列位于变异丰富的3′端而无法查到与其他物种基因的同源性。     

Repeat subtraction‐mediated sequence capture from a complex genome

Sequence capture technologies, pioneered in mammalian genomes, enable the resequencing of targeted genomic regions. Most capture protocols require blocking DNA, the production of which in large quantities can prove challenging. A blocker‐free, two‐stage capture protocol was developed using NimbleGen arrays. The first capture depletes the library of repetitive sequences, while the second enriches for target loci. This strategy was used to resequence non‐repetitive portions of an approximately 2.2 Mb chromosomal interval and a set of 43 genes dispersed in the 2.3 Gb maize genome. This approach achieved approximately 1800–3000‐fold enrichment and 80–98% coverage of targeted bases. More than 2500 SNPs were identified in target genes. Low rates of false‐positive SNP predictions were obtained, even in the presence of captured paralogous sequences. Importantly, it was possible to recover novel sequences from non‐reference alleles. The ability to design novel repeat‐subtraction and target capture arrays makes this technology accessible in any species.     

Chromosomal DNA from both flagellate and non-flagellate Bordetella species contains sequences homologous to the Salmonella H1 flagellin gene

Abstract The genus Bordetella contains four species: two are non-motile, the human pathogens B. pertussis and B. parapertussis ; and two are motile, the broad host-range mammalian pathogen B. bronchiseptica , and the avian pathogen B. avium . The motility of the latter two species is due to peritrichous flagella. Here we show that strains of all four species contain DNA sequences homologous to flagellin genes. Two types of gene probe were hybridised to Bordetella chromosomal DNA in Southern blots: the structural gene for H1 flagellin of Salmonella typhimurium and an oligonucleotide derived from the conserved N-terminal amino acid sequences of various flagellin proteins. Cla I-digested DNA from all four Bordetella species hybridised with both probes in Southern blots, although each species gave a characteristic pattern of hybridisation. This indicates that the non-motile B. pertussis and B. parapertussis species contain non-expressed flagellin genes.     

Measurement of cytochrome P4501A induction in dab (Limanda limanda) and other teleosts with species-specific cDNA probes: isolation and characterisation of dab cDNA and its use in expression studies with β-naphthoflavone-treated fish

Induction of cytochrome P4501A (CYP1A) in fish is an important biomarker in marine monitoring programmes but a number of factors complicate interpretation of data based on catalytic activity. To provide additional analytical tools, we have cloned and sequenced entire (dab) and partial cDNAs (flounder, turbot, sand eel) from several fish species. A detailed analysis comparing the new sequences to those on the database (13 sequences) is presented and identifies an invariant, teleost-specific sequence (195-IVVSVANVICGMCFGRRYDH-214) which might be the basis for production of a species cross-reactive antibody. Northern and slot blots of fish RNA (sand eel, plaice, turbot, flounder and dab) showed extensive cross-species hybridisation with each of the cDNAs (sand eel, plaice, turbot, flounder and dab). The exception was turbot RNA, which only gave adequate hybridisation when the turbot probe was used. Attempts to normalise the hybridisation data to GAPDH mRNA were not satisfactory since there were significant species differences in expression of this gene and expression was suppressed (20–40%) by β-naphthoflavone treatment. The CYP1A probes indicated induction levels relative to untreated dab of: plaice (five-fold); turbot (12-fold); flounder (12-fold); and dab (10-fold). The study demonstrates the relative ease with which species-specific molecular probes can be generated and used.     

Identification of new transposable genetic elements in Burkholderia pseudomallei using subtractive hybridisation

A subtraction library of Burkholderia pseudomallei was constructed by subtractive hybridisation of B. pseudomallei genomic DNA with Burkholderia thailandensis genomic DNA. Two clones were found to have significant sequence similarity to insertion sequences which have previously not been found in B. pseudomallei (designated ISA and ISB); and two clones showed sequence similarity to different regions of Burkholderia cepacia IS407 that has recently been detected in B. pseudomallei. The former, though possibly non-functional, represents new transposable genetic elements of B. pseudomallei. All three sequences were found to be present in multi-copy in the genomes of a number of B. pseudomallei strains and in B. thailandensis, which are the first transposable elements identified in this species.     

Isolation and Identification of Submergence-induced Genes in Maize Seedlings by Suppression Subtractive Hybridization

To investigate the expression profile of maize genes induced by submergence, a subtracted cDNA library of maize seedling roots was constructed using suppression subtractive hybridization (SSH). The cDNA of maize seedling roots treated with submergence (ST) was used as tester and what from untreated roots (UT) as driver. Products of the secondary PCR from the forward subtraction were cloned into T/A vector and transferred into Escherichia coli strain JM10B by electroporation. Four hundred and eight randomly chosen transformants carrying cDNA fragments were screened with PCR-Select Deferential Screening Kit. One hundred and eighty-four cDNA clones were identified as submergence specifically induced or highly expressed. After sequencing and removing redundant cDNAs, we got 95 submergence-induced cDNA clones. Of the 95 cDNA clones, 68 contain the regions with 60%-90% identity to their homolog in GenBank, 21 are expected to be novel genes, only 6 correspond to the published maize sequences. Key words: maize; expression profile; suppression subtractive hybridization (SSH); submergence     

Mouse histocompatibility-related genes are not conserved in other mammals

In addition to the genes for classical H-2 antigens, the H-2 complex of the mouse contains numerous homologous genes belonging to several distinct families. It is not known whether they have any functions. To address this question, I have investigated whether these genes are separately conserved in evolution. Subcloned 5' gene segments, encoding the variable domains, were used as hybridisation probes on genomic DNA blots of various mammals. Only the largest gene family, which includes the classical H-2/HLA genes, is detectable in humans and other mammals. The other gene families, including Qa-2 and T1a, are not conserved even in rodents. Most or all of their coding sequences are therefore redundant.