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.     

Structure and expression of the human creatine kinase B gene

Various cDNAs for creatine kinase type B (CK-B) were isolated from human cDNA libraries using a 26-oligonucleotide guess-mer probe. One of the cDNAs appeared to be almost full-length and contained an open reading frame coding for the 381 amino acid residues of the human CK-B polypeptide. The nucleotide sequences of the translated region as well as the primary protein structure show a high degree of homology with known CK-B and CK-M sequences of other vertebrates. The level of CK-B RNA as a measure of CK-B gene activity was determined in various human tissues and cultured cells. Our results confirm that CK-B is expressed in a tissue-specific manner and give support to the previously proposed relation between CK-B gene activity and cell proliferation. Screening of genomic DNA with various cDNA regions as probes revealed that there is only one CK-B gene per haploid genome. Gene cloning and sequencing indicated that CK-B is coded for by a relatively small gene of 3.2 kb in size, which is partially overlapped by an HTF island (A. P. Bird (1986) Nature (London) 321, 557-558) with an extremely high G + C content at its 5' end.     

Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization

Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.     

Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.     

cDNA捕捉法获得水稻杂种不育基因座位Sc区域的编码序列

摘要：cDNA捕获法足一种以表达为基础的基因分离技术,直接用目的区域的基因DNA捕捉该区域编码的cDNA,快速从大的基因组区域分离表达序列。本研究用一个水稻杂种不育基因座位Sc附近的大片段TAC基因组片段来捕捉该区域在水稻穗部表达的cDNA,共获得了6条不同的cDNA。将这些cDNA克隆进行测序分析,获得了该区域在水稻部表达的部分基因,其中1个是籼稻特有的基因。这些cDNA片段可成为新的标记用于目的基因的精细定位和候选基因序列分析。     

Generation of full-length cDNA libraries enriched for differentially expressed genes for functional genomics

Here, we describe the application of a RecA-based cloning technology to generate full-length cDNA libraries enriched for genes that are differentially expressed between tumor and normal tissue samples. First, we show that the RecA-based method can be used to enrich cDNA libraries for several target genes in a single reaction. Then, we demonstrate that this method can be extended to enrich a cDNA library for many full-length cDNA clones using fragments derived from a subtracted cDNA population. The results of these studies show that this RecA-mediated cloning technology can be used to convert subtracted cDNAs or a mixture of several cDNA fragments corresponding to differentially expressed genes into a full-length library in a single reaction. This procedure yields a population of expression-ready clones that can be used for further high-throughput functional screening.     

Expression sequence tag-specific full-length cDNA cloning: actin cDNAs

Current strategies for cDNA cloning are based on construction of cDNA libraries and colony screening. The process of obtaining a full-length cDNA clone can be highly time and labor intensive. Using the human actin gene as a model target cDNA, we have developed an RNA-capture method for rapid cloning of full-length cDNAs. The approach involves the capture of mRNA with expressed sequence tag (EST)-derived, biotin labeled antisense "capture" primers and streptavidin-coated magnetic beads. Full-length cDNA is then synthesized from purified EST-specific mRNA and cloned directly into plasmid vectors. The results of using beta-actin-based capture primers on cytoplasmic RNA were the isolation of both beta- and gamma-actin cDNA clones. Of the 16 actin-specific cDNA clones analyzed, 15 (93%) were full-length. This approach for cloning full-length cDNAs from available ESTs or partial cDNA sequences will facilitate a more rapid and efficient characterization of gene structure and function.     

柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

为了分离鉴定柔嫩艾美耳球虫(Eimeria tenella)孢子发育阶段虫体的差异表达基因,分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组,或未孢子化卵囊为驱动组、孢子化卵囊为实验组,利用抑制性消减杂交(SSH)技术,构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%,孢子化卵囊cDNA消减文库的重组率为98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从孢子化卵囊cDNA消减文库中获得了13个单一有效序列,其中8个EST与已知蛋白同源性很高;从2个子孢子cDNA消减文库中共获得了40个单一有效序列,其中9个EST与已知蛋白同源,其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。     

Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis.

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.     

A rapid method for cloning and sequencing variable-region genes of expressed immunoglobulins

A rapid procedure is described for cloning immunoglobulin V region genes from cells that express them. cDNA is synthesized from mRNA template using primers homologous to the immunoglobulin constant-region genes. Blunt-ended, double-stranded cDNA is obtained by sequential addition of enzymes to a single tube. The cDNA is inserted directly into the M13 vector, which is screened by plaque lifting for the presence of specific inserts. Screening probes can be generated from 32P-labeled single-stranded cDNAs generated from primers different from those used for cloning, or alternatively, from previously cloned V or C gene segments. The ease of cloning a cDNA V region is directly related to the abundance of Ig-specific mRNA within the cell of interest. This method minimizes the number of steps and the time needed to obtain accurate and complete sequences of any expressed Ig V region gene.     

GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs

Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cystein in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.     

用改进的差别显示法克隆R6大鼠细胞的癌相关基因

为了识别参与细胞癌变的基因,本文选择p53135-val-过表达正常细胞系R6#13-8及其自发转化癌变细胞系T2作为研究材料,在克隆差异表达基因的同时,建立了一种简单、快速的差异表达基因分离的方法,并已克隆到2个与细胞癌变相关的候选新基因。本文所得的实验结果表明这种方法实验步骤简单,避免使用同位素,RT-PCR扩增带的重复性好,能克隆到大于500bp的差异表达cDNA片段,差异表达的PCRcDNA片段假阳性率低,并可广泛适用于在两个或两个以上相对应真核细胞RNA群体中分离和克隆特异表达基因。研究结果还提示在R6#13-8自发转化为T2的过程中涉及多个基因的激活与失活,这两个细胞系之间存在明显的基因表达差异。     

Cloning of cancer-related genes of Rat6 fibroblasts by using an improved differential display method]

The p53 gene is the most frequently mutated gene identified so far in human cancers. When a mutant p53(135)-val gene was allowed to be over-expressed in Rat6(R6) cells, a high incidence of spontaneous transformation was observed in long-term culture of this cell line(R6 # 13-8). To identify genes involved in cell transformation, parental p53 over-expressing cell, R6 # 13-8, and its spontaneous transformant T2, were analyzed by an improved mRNA differential display technique, which was reproducible, simpler, and was able to clone cDNA longer than 500 bp, and was with less false positives. When 33 10-mer or 12-mer single primers with arbitrary but defined sequence were used for PCR, over 90 discrete cDNAs were obtained from R6 # 13-8 and T2 cells. Three differentially expressed cDNAs were identified, one of them is highly expressed in T2 cells, while the other two, 0.8 kb and 0.9 kb long, are highly expressed in R6 # 13-8 cells. The latter were cloned and confirmed by Northern hybridization. Both cloned fragment were not homologous with any published sequence. Our results suggest that the activation and inactivation of genes are involved in the process of the spontaneous transformation from R6 # 13-8 to T2.