Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA

This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5′ end of a gene, followed by denaturation and polyadenylation of its free 3′ ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3′ end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification.     

降低mRNA差异显示技术假阳性率的一种方法

为了探讨降低mRNA差异显示技术假阳性率的方法 ,进一步提高此技术的可靠性 ,提取了手术切除肝癌及非癌肝组织成对标本的总RNA ,逆转录获得cDNA片段 ,以mRNA差异显示方法筛选差异表达基因 ,选取较明显的一条差异表达条带 ,行进一步PCR扩增 .分别对PCR产物及其经TA克隆后随机挑选的 6个单克隆质粒DNA进行序列分析 ,并通过GenBank BLAST数据库进行序列的同源性比较 ,以Northern杂交予以来源确认 .自 72 0余条扩增条带中共选出 2 8条差异条带 .序列分析及同源性比较表明 ,所选择条带的PCR产物为一可能的新基因片段 ;而随机选择的 6个TA克隆质粒DNA中 ,有 4个为同一已知基因片段 ,一个为另一已知基因片段 ,一个为一可能的新基因片段 .同源性比较表明 ,PCR产物直接测序所得序列与TA克隆质粒DNA的 6个片段不具同源性 .结果表明 ,mRNA差异显示条带可能由 1条以上分子量相似的片段构成 ,直接对PCR产物行序列分析并以其为探针进行Northern杂交 ,是导致出现假阳性片段的原因之一 .将PCR产物进行TA克隆 ,对单克隆质粒DNA进行序列分析并以其为探针进行Northern杂交 ,可能是解决此问题的一种较好方法 .     

玉米雄性不育细胞质特异DNA片段的发现及差异展示

利用3对线粒体引物对玉米同核异质和同质异核不育系的基因组总DNA进行PCR扩增;对检测到多态性的引物,再分别对供试材料小孢子发育至四分体、单核期和双核期的花药总RNA进行差异显示分析。结果表明：以基因组总DNA为模板,引物P1-P2在所有供试不育材料都有一相同的特异扩增带,而在保持系中均无扩增;引物P3-P4在所有供试材料中均无扩增;引物P5-P6仅在保持系黄早四中有扩增,而在其他供试材料中无扩增。这一结果说明以P1-P2为引物所检测到的特异扩增带为所有供试不育细胞质所特有,且不受供试材料不同核背景的影响。对于在不育材料基因组总DNA中具有特异扩增的引物P1-P2,进一步以cDNA为模板进行PCR扩增（RT-PCR）,所有不育材料在小孢子发育的3个时期均有一相同的特异扩增带,而保持系在小孢子发育的相应时期均无扩增,说明以P1-P2为引物所检测到的转录本的大小和数目,在同核异质及同质异核不育材料间均表现一致,且不受小孢子发育时期的影响。这说明以P1-P2为引物所检测到的不育材料DNA水平的共同结构特点在小孢子发育中具有转录上的一致性,因此可以认为供试不育细胞质DNA水平的这一特异序列结构与雄性不育性状的表现有关。     

Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase

A cDNA clone encoding a monofunctional aspartate kinase (AK, ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe. Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A. thaliana bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH). By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC). The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue. The presence of two homologous genes in A. thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp. 287–294 [this issue]). This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA. The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product.     

Cloning and Expression of Small cDNA Fragment Encoding Strong Antiviral Peptide from <Emphasis Type="Italic">Celosia cristata</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A small cDNA fragment containing a ribosome-inactivating site was isolated from the leaf cDNA population of Celosia cristata by polymerase chain reaction (PCR). PCR was conducted linearly using a degenerate primer designed from the partially conserved peptide of ribosome-inactivating/antiviral proteins. Sequence analysis showed that it is 150 bp in length. The cDNA fragment was then cloned in a bacterial expression vector and expressed in Escherichia coli as a ~57 kD fused protein, and its presence was further confirmed by Western blot analysis. The recombinant protein was purified by affinity chromatography. The purified product showed strong antiviral activity towards tobacco mosaic virus on host plant leaves, Nicotiana glutinosa, indicating the presence of a putative antiviral determinant in the isolated cDNA product. It is speculated that antiviral site is at, or is separate but very close to, the ribosome-inactivating site. We nominate this short cDNA fragment reported here as a good candidate to investigate further the location of the antiviral determinants. The isolated cDNA sequence was submitted to EMBL databases under accession number of AJ535714.     

PCR检定OSM cDNA转染细胞中基因组整合与转录

用PCR和RT-PCR方法对人OSM cDNA转染的小鼠黑色素瘤细胞进行基因组整合和mRNA转录的检定.基因组整合检定时,采用与调控序列和cDNA序列相对应的上、下游引物,以连续的转录单位进行扩增,能够更准确地反映整合与表达的关系;mRNA检定时,采用与cDNA序列和质粒克隆位点与加polyA信号之间序列相对应的上、下游引物,可以区分宿主细胞中内源性与外源性基因的转录.     

Control of expression of a gene encoding an extensin by phytochrome and a blue light receptor in spores of Adiantum capillus-veneris L.

In the present study, using a newly developed fluorescent differential display technique, we have carried out large-scale screening for genes whose expression was regulated by phytochrome and antagonistically by a blue light receptor in the spores of the fern Adiantum capillus-veneris L. Spores after imbibition were briefly irradiated with red, red/blue or blue light and collected 8 h after the irradiation. Total RNA was isolated from each sample and used to make cDNA with an oligo-dT primer. The cDNA was then used as a template for PCR with the oligo-dT primer and 80 arbitrary primers. The resulting PCR products were analyzed by an automated fluorescent DNA sequencer. Among 8000 displayed bands, we identified 15 upregulated and four down-regulated bands by red light, and this red light effect was irreversibly reversed by blue light. We cloned one of the up-regulated cDNA fragments and used it to screen a cDNA library prepared from the spores. The isolated insert is predicted to encode Ser-(Pro) _n repeats and showed homology with cell wall-associated extensins. The expression of this cDNA was induced 8 h after a red light treatment and the red light induction was photoreversibly prevented by far-red light and photo-irreversibly by blue light. The mRNA of this gene was detectable 4 h after red light irradiation and gradually increased in germinating spores.     

应用改良的mRNA差异展示法克隆人鼻咽上皮特异性表达的cDNA片段

本文对ｍＲＮＡ差异展示法进行了改良．利用一组随机引物与来源于ｍＲＮＡ翻译起始位点区域的引物组合进行ＲＴ－ＰＣＲ,使差异展示的片段来源于蛋白编码区,并对差异展示的条件进行了优化．应用此法对人鼻咽上皮与软腭口腔粘膜上皮的基因表达进行了比较研究,得到了１０个在鼻咽上皮特异表达的ｃＤＮＡ片段,并对其中的５个片段进行了亚克隆和序列分析．通过与ＧｅｎｅＢａｎｋ数据库中的序列进行同源性比较,确定其中两个片段为未知新序列,Ｎｏｒｔｈｅｒｎ杂交证实其中一个片段ＮＥＳ１为鼻咽上皮特异性表达片段．     

mRNA差别显示技术分离草菇低温诱导基因*

本文报道采用改良的mRNA差别筛选法,通过选用3'锚定引物和18-25碱基的PCR随机引物组合,以琼脂糖凝胶电泳,EB显色替代聚丙烯酰胺凝胶电泳和放射自显影,对低温处理草菇(Volvariella volvacea)及正常草菇基因表达进行筛选、分析,结合RNA斑点杂交技术加以验证,分离得到70个草菇低温诱导DNA片段。     

mRNA差别显示技术分离草菇低温诱导基因

本文报道采用改良的mRNA差别筛选法,通过选用3'锚定引物和18-25碱基的PCR随机引物组合,以琼脂糖凝胶电泳,EB显色替代聚丙烯酰胺凝胶电泳和放射自显影,对低温处理草菇(Volvariella volvacea)及正常草菇基因表达进行筛选、分析,结合RNA斑点杂交技术加以验证,分离得到70个草菇低温诱导DNA片段。     

A modified procedure for PCR-based differential display and demonstration of use in plants for isolation of genes related to fruit ripening

We have modified and optimized PCR-based differential display for efficient identification and isolation of genes whose expression patterns are correlated with changes in growth, development, physiology, and/or environmental response. This protocol is general in nature and can be applied for analysis of virtually any plant tissues from which several μg of total RNA can be extracted. We report here the use of tomato fruit ripening as a model system in which to test and optimize differential display in plants. Specifically, mRNA from ripe, early breaker, mature green, and ethylene-treated mature green tomato fruit were examined to identify and distinguish non-ethylene-inducible from ethylene-inducible genes related to ripening. DNA-free total RNA was utilized as template for synthesis of first-strand complementary DNA using each of 12 possible 5′-T₁₁ XY-3′ anchor primers (X=A, C, or G; Y=A, C, G, or T). PCR amplification products of the resulting cDNA populations were generated via use of random primers in combination with the corresponding anchor primer employed for cDNA synthesis. We demonstrate that degenerative anchor primers are useful for making representative cDNA populations, but are not effective for representative display-PCR. cDNA, resulting from degenerative anchor primer synthesis, yielded substantially fewer ripening-related display-PCR products when amplified with the same degenerative anchor primer employed in cDNA synthesis, versus the corresponding set of specific anchor primers. Amplification products specific to ripe fruit cDNA were isolated directly from display gels, reamplified, cloned, and confirmed for ripening-related gene expression via RNA gel-blot analysis.     

An abnormally long HIV-1 env DNA PCR product due to altered sequences of primer binding sites

To investigate the generation of an abnormally long HIV-1 env PCR DNA product the latter was cloned and sequenced followed by sequence analysis of HIV-1 primer binding sites. We found that the formation of an abnormally long PCR product was due to HIV-1 env sequence alteration (a) in the reverse primer binding site resulting in faulty primer binding and (b) downstream from the forward primer sequence resulting in a new binding site with reverse complementary sequence with respect to the forward primer at the opposite end of the PCR product. Both changes led to amplification of a longer PCR product with forward primer alone. Our results indicate that the HIV-1 genetic diversity in the env gene can lead to amplification of a specific PCR product of unexpected size which can be disregarded in the absence of its cross-validation.     

大菱鲆肌肉组织中的基因差异表达与体长性状的相关性研究

为了揭示出一些影响鱼类生长发育速度方面的遗传信息,本文应用mRNA差异显示技术,以同一批受精卵孵化的同池养殖的两组大菱鲆鱼为试验材料,检测了在相同生长发育条件下两组体长相差悬殊的3月龄大菱鲆肌肉组织的基因差异表达。结果：18对引物组合共显示出723条带,其中有527条带能够重现,差异显示条带的重现率为72.89%;在527条稳定的条带中有21条为差异带,其余506条为共有带（96.02%）。21条差异表达条带中有16条（3.04%）为阳性差异表达cDNA片段。这些差异表达cDNA片段的存在,说明体长差异悬殊的两组大菱鲆之间存在基因表达上的差异。试验结果对于进一步分析各种差异表达基因与大菱鲆的生长性状之间的相关关系奠定了基础;为深入研究大菱鲆的生长发育性状的分子遗传机制奠定了基础。     

限制性酶切片段差异显示及其应用

差异显示技术(DD)-PCR是一种研究基因表达差异的重要而应用广泛的方法,传统的差异显示法由于在PCR时采用Poly（T）引物和随机引物而导致较高的假阳性率和产物的近Poly(A)非编码区的大量扩增。改进后的限制性酶切片段差异显示技术(RFDD)-PCR采用ToqI酶切双链cDNA,连上特殊设计的接头,再用经特殊设计的特异性配对于接头的引物来扩增,因此能重点扩增编码区并能极大地消除假阳性率。由于扩增时引物就带有荧光或放射性核素标记,还使得差异显示条带的检测更为方便、灵敏。本简要介绍了该法的原理、步骤、应用及其优缺点。