A general method for detection and characterization of an mRNA using an oligonucleotide probe

A general method for the detection and characterization of an mRNA using an oligodeoxynucleotide probe is described. The results presented indicate that a G-dT or a dG-U base pair within a short DNA-RNA hybrid does not significantly reduce the stability of the hybrid. On this basis, we propose that 11 amino acids, including Trp and Met, can be used in selecting a peptide sequence suitable for use in designing an oligodeoxynucleotide probe complementary to a given mRNA. To test this hypothesis, we have synthesized an oligodeoxynucleotide (oligo II) complementary to the region of gastrin mRNA coding for Trp-Met-Asp-Phe and have compared its effectiveness as a hybridization probe and as a primer for the synthesis of gastrin-specific cDNA with another oligonucleotide (oligo I) complementary to the region of gastrin mRNA coding for Trp-Met-Glu-Glu. Unlike oligo I, oligo II functions as a primer in specific cDNA synthesis only when the mRNA is denatured prior to initiation of synthesis. Similarly, oligo II can be used as a specific hybridization probe for gastrin mRNA only when the RNA is denatured and partially cleaved with NaOH before hybridization. A simple procedure for denaturing gastrin mRNA to ensure efficient interaction with oligodeoxynucleotide probes is described. This procedure should be useful in studies with other oligonucleotides and mRNAs as well.     

A hybridization and enzyme blocking method to enrich minor cDNA sequences

A method is presented which allows for the enrichment of low frequency cDNA sequences. The crucial step in the procedure is the hybridization of a pool of cDNA to homologous or heterologous RNA to a Rot value which will leave minor sequences in a single strand cDNA form while the major sequences form cDNA:RNA hybrids. This allows subsequent enzymatic differentiation between major and minor sequences resulting ultimately in the degradation of the major sequences. The procedure is general and simple as it requires no column chromatography step. The method is designed to integrate into a widely used cDNA cloning protocol and results either in double-stranded cDNA which can be used for molecular cloning or as a source of probes for hybridization.     

Locus-specific vector/primer systems for rapid cloning of allelic variants

We have developed a rapid cDNA cloning procedure which uses a single-stranded (ss) vector/primer in which the primer sequence is locus-specific. Vector/primers were constructed by substituting a specific oligodeoxynucleotide primer sequence in place of the polylinker in M13mp19. The ss vector/primer is linearized and used to prime cDNA synthesis. Recircularized DNA is then used directly to transform competent bacterial hosts. As no intermediate column purifications or extractions are necessary, the entire procedure is performed in a single tube, contributing to the overall simplicity of the protocol. The primary use for this kind of vector/primer system will be for cloning and sequencing multiple allelic variants of polymorphic loci which contain a conserved 3' sequence. The two vector/primers we report here are specific for HLA-DQ beta genes and for human Ig variable regions associated with IgM antibodies.     

mRNA quantitation by a simple and sensitive RNAse protection assay.

An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.     

大肠杆菌表达质粒pSM43及pSM53的构建

利用已成功高表达ｅｒａ基因的质粒ｐＣＥ３１翻译起始码上游的序列,去构建大肠杆菌新的外源基因表达载体。先合成特定序列的单链脱氧寡核苷酸,以改进的实验程序插入ｐＪＬ６,其后再加上限制酶多克隆位点。所构建的ｐＳＭ４３和ｐＳＭ５３分别适合於不带翻译起始码（ＡＴＧ）和带起始码的基因插入、表达非融合目的蛋白质之用。并已成功用於人肿瘤坏死因子、人骨形成蛋白、ＨＩＶ蛋白酶、Ｄｕｃｈｅｎｎｅ肌营养不良等ｃＤＮＡ基因的高表达。     

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning

We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the lambda EXLX(+) and lambda EXLX(-) vectors that can be used for the expression in Escherichia coli of proteins encoded by cDNA inserts. This is achieved by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the lambda SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage lambda to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage lambda arms, lambda LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of a specialized plasmid between these sites will convert it into a phage lambda cDNA cloning vector with automatic plasmid subcloning capability.     

A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free (sup degrees) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (-)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup degrees host we promote the propagation of heteroduplexes with the oligo-containing (-)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2-20% for single base changes and from 0.3-9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (-)strands.     

cDNA selection with YACs

Identification of expressed sequence tags (ESTs) in large genomic segments is an important step in positional cloning and genomic mapping studies. A simple and efficient polymerase chain reaction (PCR)-based approach is described here to identify coding sequences in large genomic fragments of DNA cloned in vectors such as yeast artificial chromosome (YAC) vectors. The method is based on blocking of sequences such as repetitive and GC rich sequences in the genomic DNA immobilized on nylon paper discs prior to hybridization of the discs to cDNA library, and recovery of the selected cDNAs by the PCR. Single or multiple cDNA libraries can be used in the selection procedure. The procedure has been used successfully also with total yeast DNA containing a YAC.     

SMART技术构建衣藻细胞的酵母双杂交文库

衣藻是用来研究植物光合作用和动物纤毛常用的模式生物.为了研究蛋白质之间的相互作用,利用SMART技术构建了衣藻的酵母双杂交文库.使用Trizol试剂提取鞭毛再生过程和光周期培养的细胞进入分裂前的衣藻细胞的总RNA,经过Oligotex纯化得到mRNA;应用SMART技术和LD-PCR合成双链cDNA,经过CHROMA SPIN TE-400柱子去除短片段的cDNA;cDNA和线性化载体pGADT7-Rec共转化酵母Y187构建酵母双杂交文库.库容达到3.0 × 106CFU,重组率为70％,插入片段平均长度为0.6 kb.以上结果说明该文库质量较好,能够通过筛选文库得到与目的蛋白互相作用的蛋白质,为寻找蛋白质的作用伴侣打下基础.     

三丁基锡暴露条件下杂色鲍肝胰腺均一化cDNA文库的构建

用RDP试剂提取三丁基锡暴露诱导的杂色鲍肝胰腺总RNA,经Oligotex纯化得到 mRNA;应用SMART技术合成双链cDNA,双链特异核酸酶(DSN)进行双链cDNA的均一化,构建了杂色鲍三丁基锡暴露诱导下的均一化cDNA文库.原始文库的库容为4.3× 106 CFU/ml,重组率为 97.9%.从文库中随机挑选了3 288个克隆进行测序,得到3 048个高质量EST序列,其中有 370条 Contigs,2 103条 Singlets,Unigenes共 2 473条,冗余率为18.86%.以上结果说明该文库质量较好,为进一步筛选相关功能基因打下基础;较低的冗余率说明该文库值得继续使用大规模ESTs测序的方法寻找相关功能基因,并为进一步使用基因芯片技术研究相关功能基因的表达谱提供便利.     

A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector.

cDNA expression cloning is a powerful method for the rescue and identification of genes that are able to confer a readily identifiable phenotype on specific cell types. Retroviral vectors provide several advantages over DNA-mediated gene transfer for the introduction of expression libraries into eukaryotic cells since they can be used to express genes in a wide range of cell types, including those that form important experimental systems such as the hemopoietic system. We describe here a straightforward and efficient method for generating expression libraries by using a murine retroviral vector. Essentially, the method involves the directional cloning of cDNA into the retroviral vector and the generation of pools of stable ecotropic virus producing cells from this DNA. The cells so derived constitute the library, and the virus they yield is used to infect appropriate target cells for subsequent functional screening. We have demonstrated the feasibility of this procedure by constructing several large retroviral libraries (10(5) to 10(6) individual clones) and then using one of these libraries to isolate cDNAs for interleukin-3 and granulocyte-macrophage colony-stimulating factor on the basis of the ability of these factors to confer autonomous growth on the factor-dependent hemopoietic cell line FDC-P1. Moreover, the frequency at which these factor-independent clones were isolated approximated the frequency at which they were represented in the original plasmid library. These results suggest that expression cloning with retroviruses is a practical and efficient procedure and should be a valuable method for the isolation of important regulatory genes.     

Use of the polymerase chain reaction for the differential cross screening of libraries cloned into phage-lambda-based vectors

We describe a simple and rapid method that can be used to identify sequences present in any two DNA libraries (either genomic or cDNA), provided only that the libraries are in different vectors with different cloning sites. This procedure makes use of the polymerase chain reaction (PCR) to amplify the inserts of one library. The product of the PCR reaction is then used to screen a second library to identify sequences which are common to both. We illustrate the use of this method for the systematic isolation of human X-chromosome-linked genomic clones that harbor sequences expressed in human chorioretinal tissue.     

Screening recombinant phage M13 plaques with RNA probes; a one-step procedure which identifies clones containing either of the complementary DNA strands

We describe a method for detecting specific DNA sequences cloned in M13 phage vectors, based on the procedure of Woo (in Wu, R., Methods in Enzymology, Vol. 68, Academic Press, New York, 1979, pp. 389-395). M13 plaques are adsorbed to a nitrocellulose filter that has been pre-saturated with bacteria. The filter is incubated on an agar plate to amplify the phage; the DNA is alkali-denatured and then hybridized with a radioactive RNA probe. Unlike standard procedures, this method detects and distinguishes M13 plaques containing phage particles which harbor either the coding or non-coding (RNA-like) DNA strand, when single-stranded RNA is used as probe. We have optimized this procedure with M13 clones containing mouse histidine tRNA gene sequences and have used it to determine the sequence of both strands of a mouse glycine tRNA gene.     

烟草花叶病毒运动蛋白基因的cDNA克隆、序列测定及植物转化

植物病毒侵染宿主植物的一个重要过程是通过它在宿主体内的转移和传播,产生病害。植物病毒在宿主体内的转移主要有两种方式,一种是通过植物维管组织进行的系统转移,另一种是植物病毒在宿主细胞之间的转移,这种转移是通过植物细胞的胞间连丝实现的。实验表明,病毒自身编码的一种蛋白参与了这个转移过程,对烟草花叶病毒(TMV)而言,这种蛋白就是分子量为30kDa的运动蛋白。