cDNA cloning by amplification of circularized first strand cDNAs reveals non-IRE-regulated iron-responsive mRNAs

Currently, the rapid amplification of cDNA ends (RACE) is the most common method for PCR cloning of cDNA. Because RACE uses a gene specific primer and one adaptor primer that is shared by all cDNAs may result in numerous nonspecific products that can hinder the cloning process. Here we report a new method that uses circularized first strand cDNA from mRNA and two gene specific primers to amplify both the 5' and 3' cDNA ends in one reaction. A cDNA band of correct size can be obtained on the first pass in this approach. If the correct size is not obtained on the first pass, amplification of cDNA ends can be repeated until the correct size of the cDNA is obtained. We tested this new method on eight mRNAs that we have previously shown to respond to cellular iron levels. We obtained sequences for six mRNAs that were 43 bp to 1324 bp longer than that reported in GenBank and obtained the same length sequence for the other two mRNAs. RNA folding program shows no iron responsive elements (IRE) on these mRNA. In conclusion, our cloning approach offers a more efficient method for cloning full-length cDNA and it may be used to replace the existing method of 5' end cDNA extension. The data enabled us to exclude the possibility that the expression of these iron responsive genes are regulated by IREs.     

A simple method for obtaining cell-specific cDNA from small numbers of growing root-hair cells in Arabidopsis thaliana

A simple and rapid method for cloning specific cDNAs from mRNA populations derived solely from small numbers of root-hair cells is described here. To identify genes expressed during the earliest visible stage of root-hair cell development, cell contents were aspirated from small numbers of Arabidopsis root-hair cells at or just before this stage. This material was used to make reusable solid-phase oligo-dT-primed cDNA libraries. To demonstrate that the libraries contained high quality longer cDNAs, a fragment located 2.7 kb from the 3' end of the cDNA of the single copy root-hair expressed gene RHD3 was cloned using a nested PCR strategy. This technique was also used to obtain novel gene expression information by cloning the full-length 0.85 kb cDNA of the Rop2 GTPase from this library. This approach offers a means of cloning larger cDNAs directly from small numbers of growing root-hair cells and, potentially, other epidermal cell types.     

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.     

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.     

Cloning and expression of cynomolgus monkey and baboon zona pellucida proteins

Partial clones for the three cynomolgus monkey (Macaca fasicularis) zona pellucida genes (cmZPA, cmZPB, and cmZPC) have previously been isolated. These partial clones contained the sequences for the C-terminal portion of each rcmZP protein. To obtain full-length clones for each cmZP, a fresh cynomolgus monkey ovarian cDNA library was constructed. PCR methodology was employed to speed the isolation of full-length clones for each cmZP cDNA. The 3' primers were designed based on sequence information from the previously identified clones; the 5' primers were designed using the human ZP sequences. The PCR technique yielded full-length clones of cmZPA and cmZPC, but not of cmZPB. Therefore, a genomic clone of cmZPB was isolated and the sequence determined. The exon/intron structure is nearly identical to the human ZPB exon/intron structure. New PCR primers were designed based on the cynomolgus monkey ZPB genomic sequence, and a full-length cmZPB cDNA was obtained. The same primers that were used to generate the cmZPB were also used to generate a baboon (Papio cynocephalus) ZPB (bZPB) cDNA. As was done previously for the human zona pellucida (hZP) cDNAs, the cmZP, and bZPB cDNAs were transferred to shuttle vectors for transfection into Chinese Hamster Ovary (CHO) cells. Stable cell lines for producing each ZP protein were isolated. Each cell line secreted the desired recombinant zona pellucida (rZP) protein into the culture medium, and each protein was purified using an established protocol. In terms of size and purity, the purified recombinant cmZP (rcmZP) and rbZPB proteins resemble the rhZP proteins.     

用基因组DNA剪接技术克隆SIgA相关基因

目的：克隆分泌型IgA（SIgA）相关基因--J链基因(IgJ)、多聚免疫球蛋白受体基因（pIgR）和IgA重链恒定区基因（IGHA）,为进一步构建SIgA真核表达质粒奠定基础。方法：采用本室建立的"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果：PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明本实验获得的上述基因与GenBank中的目标基因序列完全一致。结论：本文通过基因组DNA剪接技术成功克隆人类SIgA三个相关基因,提示此技术是合成多外显子cDNA的有效手段。     

A procedure for selective full length cDNA cloning of specific RNA species.

A method allowing routine establishment of full length and functionally competent cDNA clones of particular mRNAs from small preparations of polyadenylated RNA is described. Pairs of synthetic primers are used for first and second strand synthesis. They include sequences complementary to the 3' terminal regions of the mRNAs and of the full length first cDNA strands, respectively and bear a few additional nucleotides at their 5' ends. After synthesis of both cDNA strands in one tube, they are precisely trimmed back with T4 DNA polymerase in presence of only two nucleoside triphosphates, to yield sticky ends fitting into a vector plasmid cleaved with two restriction endonucleases. The procedure was first applied to the simultaneous cloning of all five major measles virus (MV) mRNA species from a persistently infected cell line. Two thirds of all clones contained full length MV-specific cDNAs. Screening of less than 200 clones was sufficient to obtain several independent clones corresponding to each mRNA, except for gene F which was represented only once.     

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.     

Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer

A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.     

Identification and characterization of three genes and two pseudogenes on chromosome 13

A study was conducted on the feasibility of isolating genes and pseudogenes that map to chromosome 13 by a hybridization-based approach using a 13-specific library and pools of repeat-free cDNA clones. Five pairs of cDNA and chromosome 13 genomic clones were identified and characterized. Partial or full-length sequence was derived from all cDNAs, and database searches were performed for putative gene identification. Partial sequence was also obtained from the chromosome 13 genomic clones for comparison with those of the hybridizing cDNAs. As a result of these analyses we identified three genes, a putative homologue of a porcine mRNA encoding an unidentified hepatic protein, a putative homologue of a yeast integral membrane protein, and a gene for a translationally controlled tumor protein, and two processed pseudogenes, ribosomal proteins L23a and S3a. The latter was formerly identified as the v-fos transformation effector gene, Fte-1, and recently cited as a possible candidate for the BRCA2 gene on chromosome 13. All genes and pseudogenes were localized to cytogenetic bands by in situ hybridization of metaphase chromosomes with probes derived from the chromosome 13 genomic clones.     

Sequencing and Analysis of Approximately 40 000 Soybean cDNA Clones from a Full-Length-Enriched cDNA Library

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5′ and 3′ ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5′- and 3′-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.Key words: EST, full-length cDNA, functional annotation, legume, soybean     

Characterization of the cDNA and genomic sequence of a G protein gamma subunit (gamma 5).

A cDNA from human placenta and liver tissues that contained both sequence for the lysosomal glycosidase di-N-acetylchitobiase and sequence homologous to the gamma subunit of GTP-binding proteins was previously isolated. Here we have shown that the gamma-subunit-homologous portion of this unusual cDNA is derived from a member of the gamma-subunit multigene family. The partial human gamma-subunit sequence was used to isolate the corresponding full-length cDNA clones from bovine and rat livers. The two cDNAs encode identical 68-amino-acid proteins (7.3 kDa) homologous to previously cloned G protein gamma subunits. The bovine gene sequence encoding this new gamma-subunit isoform (gamma 5) was determined and found to have an intron-exon structure consistent with the original human chitobiase-gamma 5-subunit hybrid mRNA being a product of alternative splicing. Genomic cloning also resulted in the isolation of a human gamma 5 pseudogene.     

Cloning full-length cDNA of grapevine chrome mosaic nepovirus

Full-length cDNA copies of the genomic RNAs of grapevine chrome mosaic virus were obtained and cloned in Escherichia coli by a one-step procedure. The cloning protocol included size selections by agarose-gel electrophoresis of both the single-stranded and the double-stranded full-length cDNAs. First-strand cDNA synthesis was primed with oligodeoxythymidine while second-strand synthesis was primed with specific synthetic oligodeoxynucleotides, allowing cloning of the 3' poly(A) and of the last 5' nucleotides of the viral RNA template. For the 7.2-kb and 4.4-kb viral RNAs, up to 20% and 80%, respectively, of the clones were found to be full-length. Even for large templates, this procedure allows fast and efficient cloning of full-length cDNAs.