Specific DNA Alterations Associated with the Environmental Induction of Heritable Changes in Flax

Several flax varieties have been shown to undergo environmentally induced heritable changes resulting in stable lines termed genotrophs. The most notable of these is the variety Stormont Cirrus, also termed "plastic" or Pl. A number of morphological, biochemical and genetic differences are associated with environmental induction of heritable changes in flax. We have studied 5S rDNA alterations as a model system for understanding environmental induction of heritable changes in flax. This paper reports the isolation of a flax 5S rRNA gene variant which identifies genotroph specific restriction fragment length polymorphisms (RFLPs) in flax. Restriction fragment patterns for several enzymes were observed in both large and small genotrophs which consistently differed from the progenitor, Stormont Cirrus. Identical RFLP profiles for all restriction endonucleases tested were observed in four small genotrophs produced from separate environmental induction experiments. Comparison between Stormont Cirrus and these small genotrophs showed at least six differing bands in addition to several high molecular weight polymorphisms. Genetic data indicate that the polymorphisms were all produced from a repetitive 5S rRNA gene cluster at a single chromosomal locus. Similar, but not identical, polymorphisms are also detected in other flax varieties and Linum species suggesting that the induced variation is related to that which occurs naturally. The results are evidence that a specific set of DNA alterations occur in association with the induction of heritable changes in flax. This is the first genetic marker which is altered to an identical state in one type of genotroph. The results are discussed with respect to mechanisms for environmentally induced heritable change in plants.     

银额果蝇B染色体特异性基因的初步研究

采用代表性差异分析法（RDA）研究了银额果蝇两个单雌系AKM46（含B染色体）和AGZ2（不含B染色体）两基因组间的差异。用AKM46作检测（tester）扩增子,AGZ2作驱赶（driver）扩增子,通过三轮消减杂交后,获得了6个差异片段（100bp～300bp）。亚克隆后,对11个片段测序并与GenBank数据库进行同源性比较分析,获得了9个新的序列。选择clone22及clone42进行Southern杂交分析,这两个片段仅在检测扩增子及第一、第二、第三轮差异片段中检测到杂交信号,而在驱赶扩增子检测不到杂交信号。证实了这两个片段来自含有B染色体的单雌系AKM46,而且可能是B染色体上的特异基因片段。     

Targeted mapping of rye chromatin in wheat by representational difference analysis.

A targeted mapping strategy using representational difference analysis (RDA) was employed to isolate new restriction fragment length polymorphism probes for the long arm of chromosome 6 in rye (6RL), which carries a gene for resistance to Hessian fly larvae. Fragments from the 6RL arm were specifically isolated using a 'Chinese Spring' (CS) wheat - rye ditelosomic addition line (CSDT6RL) as tester, and CS and (or) CS4R as the driver for the genomic subtraction. Three RDA experiments were performed using BamHI amplicons, two of which were successful in producing low-copy clones. All low-copy clones were confirmed to have originated from 6RL, indicating substantial enrichment for target sequences. Two mapping populations, both of which are derived from a cross between two similar wheat-rye translocation lines, were used to map five RDA probes as well as five wheat probes. One of the populations was prescreened for recombinants by C-banding analysis. Fifteen loci, including seven new RDA markers, were placed on a map of the distal half of 6RL. The Hessian fly resistance gene was localized by mapping and C-banding analysis to approximately the terminal 1% of the arm. The utility of RDA as a method of targeted mapping in cereals and prospects for map-based cloning of the resistance gene are discussed.     

Heritable effects of 5-azacytidine treatments on the growth and development of flax (Linum usitatissimum) genotrophs and genotypes.

Seed of flax (Linum usitatissimum) were treated for short durations with 5-azacytidine and the direct and heritable effects of the treatments on plant growth and development in general and, more specifically, on the contrasting phenotypes of Durrant's large and small genotrophs were examined. 5-Azacytidine induced a reduction in the height of the plants grown from treated seed. Twenty-two percent of the first generation progeny of these plants also had short phenotypes and, in most cases, the short phenotype was stably and uniformly inherited by the second generation progeny of the short, first generation plants. Treatment also induced a marked decrease in the flowering age in a few of the first generation plants that was also transmitted to their second generation progenies. The effects seen in the progeny generations suggest that most, if not all, of the heritable changes induced by the treatment are epigenetic. Several differences were seen between the large and small genotrophs, which indicate that the genome of the small genotroph is less susceptible, than the genome of the large genotroph, to 5-azacytidine induced heritable alterations.     

Representational difference analysis using minute quantities of DNA.

Representational difference analysis (RDA) is a differential hybridization method which can effectively isolate unique DNA sequences from complex and highly related genomes or cDNA libraries. A major drawback of the RDA analysis is the requirement for pure driver and relatively pure tester samples, ruling out the analysis of whole tissue biopsies. To circumvent this problem, we have modified the technique for the analysis of very small quantities of DNA so that pure cell populations isolated by micromanipulation from tissue sections can be analyzed. Using this modified technique, as few as 50 diploid cells ( approximately 500 pg of DNA) can be analyzed.     

应用代表性差异分析法筛选人癌候选抑癌基因

运用cDNA代表性差异分析法(cDNA representational difference analysis,cDNA RDA),以正常人鼻咽上皮细胞及鼻咽癌HNE1细胞作为比较的样品来源,分离了四个在鼻咽癌中缺失的cDNA片段.以此四个片段作探针,分别进行DNA杂交、RNA杂交,结果显示,这些差异性的cDNA序列确实来自正常人鼻咽上皮且只在其中表达和/或在鼻咽癌HNE1中表达降低,并在鼻咽癌病人中存在不同程度的缺失.序列分析结果表明这些差异性表达的基因为具有相当抑癌基因功能的已知基因和可能与鼻咽癌相关的抑癌基因的新基因.从而说明cDNA RDA是一种高效、敏感、假阳性低的克隆抑癌基因的有效方法.     

Identification of sulI allele of dihydropteroate synthase by representational difference analysis in Haemophilus parasuis serovar 2

AIMS: Identification of genes differentially present in Haemophilus parasuis serovar 2 by representational difference analysis (RDA). METHODS AND RESULTS: Bacterial genomic DNA was extracted, cleaved with Sau3AI and ligated to oligonucleotide adapter pair. The optimal tester (H. parasuis serovar 2)/driver ratio (H. parasuis serovars 1, 3 and 5) for the hybridization was established and the mixture was hybridized, and amplified by PCR. The products were cloned and transformed into Escherichia coli TOP10 cells and checked for specificity by Southern blotting analysis. The RDA subtractive technique yielded six bands ranging from 1500 to 200 bp, which were cloned into pCR II-TOPO vector and 40 clones were analysed. A fragment of 369 bp was specific for H. parasuis serovar 2, and showed 99% homology to sulI gene encoding for dihydropteroate synthase (dhps). The dhps gene conferring sulfonamide resistance was detected in H. parasuis serovar 2 but was absent in serovars 1, 3, 5 and in most of the Actinobacillus pleuropneumoniae serotypes (except serotype 7). CONCLUSION: sulI allele of dihydropteroate synthase has been identified in H. parasuis serovar 2 by RDA technique. SIGNIFICANCE AND IMPACT OF THE STUDY: The RDA technique seems to be an useful method for the identification of genes that are differentially present in H. parasuis, a respiratory pathogen of veterinary interest.     

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     

用RDA技术寻找肝再生相关基因的初步研究

利用表达性差异显示分析 (RDA)技术 ,研究大鼠 2 /3肝部分切除后 1h肝组织中基因的选择性表达 ,建立了大鼠 2 /3肝部分切除术后 1h再生肝组织选择性表达基因EST库。该库约含 3× 10 4 个独立克隆 ,其中 95 %以上的克隆含有插入片段 ,长度约 2 0 0～ 70 0bp不等 ,对随机挑出的 5 2个克隆的序列分析表明其中大多数基因与肝再生调控相关 (38/5 2 )。 10株未报道序列经RNA杂交证实 ,其中 6株与肝再生相关。     

Characterization of transposable elements in the genome of rice (Oryza sativa L.) using Representational Difference Analysis (RDA)

Representational Difference Analysis was applied to characterize genomic differentiations between rice ( Oryza sativa) and foxtail millet ( Setaria italica) and subsequently to identify rice transposable elements. Rice was used as the tester and millet as the driver. A total of eleven, non-redundant, positive clones were isolated from the library. Their analysis revealed that they all represent dispersed repetitive DNA sequences. In addition, homology searches using the BLAST procedure showed that they correspond to seven distinct rice transposable elements. Three had been previously identified as gypsy-like retroelements ( Retrosat1, RIRE3 and RIRE8). The remaining four are novel: we named them hipa (a CACTA-like transposon), houba (a copia-like retroelement), hopi and dagul (two gypsy-like retroelements). The RDA clones were used as probes in Southern hybridization experiments with genomic DNAs of several species from the family Poaceae. The results suggest that the genomic differentiations associated with the activity of these transposable elements are of relatively recent origin. In addition, comparison of the hybridization patterns obtained for several Oryza species suggests that several independent amplifications of these transposable elements might have occurred within the genus.     

Chromosomal and molecular analysis of 5S RNA gene organization in the flax, Linum usitatissimum

The 5S rRNA genes (5S DNA) comprise up to 3% of the flax genome. Large copy-number changes in 5S DNA have been observed in flax genotrophs. We have characterized the chromosomal and molecular organization of this large gene family. In situ hybridization studies indicate the 5S DNA is distributed over many chromosomes, unlike most plants studied to date. Eleven genomic clones were isolated and characterized. All but one of the clones contain both 5S DNA and non-5S DNA. The homology of the 5S DNA of each clone, to a previously isolated flax 5S plasmid clone (pBG13), was determined. Five groups of 5S DNA were identified based on shared identity and repeat unit size. Group-1 and group-2 clones are the most abundant in terms of genomic representation. The remaining groups are significantly different from the previously described flax 5S DNA and are in low representation in comparison to group-1 and group-2 5S DNA. The results establish the presence of several groups of 5S DNA which are distributed over many chromosomes. The extent of identity shared among these groups to pBG13, indicates a high degree of divergence between the different groups.     

Variation in the isozymes of flax (Linum usitatissimum) genotrophs

Peroxidase, esterase, and acid phosphatase isozymes of environmentally induced L and S genotrophs, nuclear DNA reversion types, and the orginal plastic (Pl) type of the flax variety Stormont Cirrus have been compared by polyacrylamide gel electrophoresis. Differences were observed in particular line was not correlated with the nuclear DNA amount. The relationship between the isozyme pattern and the phenotypes of the lines in which they are expressed is discussed.     

Segregation of the isozymes of flax genotrophs

The segregation of isozymes of peroxidase and acid phosphatase in progenies of crosses between large (L) and small (S and L₆) flax genotrophs has been determined. The peroxidase isozymes segregated as expected on a simple Mendelian model with a dominant and a recessive allele and with the L genotroph being a homozygous dominant. All the peroxidase isozymes which differed segregated together, so the isozymes are controlled by either a single locus or closely linked loci. The acid phosphatase isozymes in the F₁ were all L type, but the segregations observed in the F₂ were not always consistent with a simple Mendelian model.     

Generation of polymorphic markers tightly linked to the thymus enlargement loci by phenotype-directed representational difference analysis

To obtain genetic markers linked to a specific genetic locus, genomic subtraction with a DNA pool of backcross or F₂ intercross animals with a specific genotype at the locus is known to be effective. To determine whether the pooling strategy is also effective for isolation of genetic markers linked to a quantitative phenotype that can potentially be controlled by multiple genetic loci, we tested the ability of representational difference analysis (RDA) to isolate genetic markers linked to the thymus enlargement observed in the BUF/Mna (BUF) rat. This is known to be controlled by single major and minor genes, Ten1 and Ten2, on Chromosomes (Chrs) 1 and 13, respectively, both of which have dose effects on the normal WKY/Ncj (WKY) allele. DNA from an inbred WKY rat was used as the tester, and the driver was prepared from a DNA pool of 12 (WKY × BUF)F₁× BUF backcross rats with high thymus ratios (thymus weight/body weight), expected to have dominance of the BUF allele in the responsible loci. By two RDA series with the restriction enzymes BglII and BamHI, respectively, 28 polymorphic markers were isolated, and 8 of them were shown to be linked to Ten1, and one to Ten2. One of the 8 markers linked to Ten1 demonstrated no recombination in 18 rats with high thymus ratios. RDA with a DNA pool based on a quantitative phenotype (phenotype-directed RDA) can thus be considered an efficient approach for direct isolation of polymorphic markers linked to a quantitative trait. Received: 15 April 1997 / Accepted: 8 May 1998     

Possible posttranslational modification,and its genetic control,in flax genotroph isozymes

Environmentally induced flax genotrophs L and S show heritable shifts in the relative mobilities of peroxidase, esterase, and acid phosphatase isozymes, plus a number of nonspecific glycoproteins. All L isozymes migrated faster than corresponding S isozymes in 10% acrylamide gels. Various aspects of these shifts are reviewed here; it is proposed that posttranslational modification, probably of the carbohydrate moieties of these glycoproteins, underlies the shifts. This proposal is discussed in relation to the switch model for genotroph induction.The financial assistance of the Natural Sciences and Engineering Research Council of Canada is acknowledged with thanks.     

应用抑制性消减杂交技术克隆大鼠肝再生过程中特异表达基因

应用抑制性消减杂交技术成功地构建了高消减效率的正向消减cDNA文库,从随机挑取的50个克隆中有31个均检出了60~400bp插入片段,对这些插入cDNA片段进行测序后经Genbank同源性检索,表明其中7个片段为未知新序列。大鼠肝切除后肝再生cDNA正向消减文库的建立为进一步大批量筛选、克隆肝再生特异性表达的未知新基因奠定了基础,初步筛选出的特异性表达的序列标记为进一步研究肝再生中基因的功能提供了依据。 Abstract:The cDNA from rat regenerating liver tissue was used as the tester and that from normal liver was used as the driver.A highly efficient subtractive cDNA library was constructed by suppression subtractive hybridization(SSH).After screening,31 clones from 50 clones which were derived from the cDNA library were inserted by 60~400bp cDNA fragments.24 cDNA fragments corresponded to known genes and 7 cDNA fragments were unknown sequences(GenBank accession number:BG447490~447496).     

用抑制差减杂交法分离和克隆梭梭幼苗受渗透胁迫诱导相关基因的cDNA片段

以Hoagland溶液培养的梭梭幼苗(H)为对照群体,甘露醇处理的梭梭幼苗(M)为目标群体,进行抑制差减杂交.用经过H cDNA差减的M cDNA构建了一个含有大约400个独立克隆的差减文库;采用差减前的H cDNA和M cDNA以及正向/反向差减杂交后的cDNA为模板标记探针,对随机挑取的100个重组质粒进行差示筛选,获得了21个阳性候选克隆.从这些阳性候选克隆中随机挑取了8个进行Northern blot分析,证实其中3个候选克隆代表了在M中特异表达或表达增强的基因,序列分析和同源性比较表明它们与逆境胁迫有关;而另外5个候选克隆无Northem杂交信号,推测它们为低丰度转录本.     

Construction of the seed-coat cDNA microarray and screening of differentially expressed genes in barley

Proanthocyanidins are dimeric or polymeric conden-sation products of the flavonoids, including catechin,epicatechin or gallocatechin with leucocyanidin, leuco-pelargonidin or leucodelphinidin [1]. They are prominentcolorless compounds, and are found widely existed inthe bark of trees, leaves, fruits, flowers and seed coats.They have many natural functions, such as antioxidantproperties [2] and insect resistance [3]. In forage, theycan bind and precipitate dietary proteins, thus protectthe anim…     

小菜蛾对溴氰菊酯抗性相关的DNA片段的克隆与分析

采用代表性差异分析（representational difference analysis, RDA ）方法,以小菜蛾Plutella xylostella (L.)的敏感品系作为对照组,溴氰菊酯抗性近等基因系（near isogenic line）作为测试组,通过三轮消减杂交得到大小为 150～300 bp的差异扩增带,经亚克隆测序并与GenBank等数据库同源比较,发现差异片段与已知抗性相关基因无同源性;以随机选取的差异片段作探针进行Southern blot分析,在测试组扩增子和三轮差异产物中都获得了阳性结果,而在对照组扩增子中的结果是阴性的。这些结果表明测序片段可能是与抗性相关的新基因序列或调控序列。