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PubMed2002

Systematic sequencing of cDNA clones using the transposon Tn5.

In parallel with the production of genomic sequence data, attention is being focused on the generation of comprehensive cDNA-sequence resources. Such efforts are increasingly emphasizing the production of high-accuracy sequence corresponding to the entire insert of cDNA clones, especially those presumed to reflect the full-length mRNA. The complete sequencing of cDNA clones on a large scale presents unique challenges because of the generally small, yet heterogeneous, sizes of the cloned inserts. We have developed a strategy for high-throughput sequencing of cDNA clones using the transposon Tn5. This approach has been tailored for implementation within an existing large-scale 'shotgun-style' sequencing program, although it could be readily adapted for use in virtually any sequencing environment. In addition, we have developed a modified version of our strategy that can be applied to cDNA clones with large cloning vectors, thereby overcoming a potential limitation of transposon-based approaches. Here we describe the details of our cDNA-sequencing pipeline, including a summary of the experience in sequencing more than 4200 cDNA clones to produce more than 8 million base pairs of high-accuracy cDNA sequence. These data provide both convincing evidence that the insertion of Tn5 into cDNA clones is sufficiently random for its effective use in large-scale cDNA sequencing as well as interesting insight about the sequence context preferred for insertion by Tn5.

Base Composition↗

PubMed1991

Study of transcription in measles virus-infected Vero cells using cDNA probes prepared from poly(A)RNA from uninfected and infected cells.

From af primary plasmid cDNA library prepared from measles virus-infected Vero cell poly(A)RNA, 435 clones selected at random were used to examine the sensitivity and specificity of cDNA probes derived from total poly(A)RNA from uninfected and infected Vero cells. The correlation between the abundance level of a particular species in the cDNA probe and the hybridization signal strength generated by the corresponding cDNA clone on a filter was reliably determined only when at least three independently prepared filters were examined. Variation in the amount of target plasmid was the most important cause of spurious signals. Variation in cDNA insert length did not disturb the signal strength within certain limits. cDNA species with abundance levels down to 0.08-0.01% were able to produce a hybridization signal above background. Unspecific cross-hybridization was shown to define the sensitivity limit of mixed cDNA probes. Despite the many false signals present at different stages, cDNA probes provided valuable information: the cDNA probes were used to monitor relative RNA expression levels and to clone five different measles virus transcripts and 2 host cell transcripts more abundantly expressed in infected cells. The abundance levels of the measles virus nucleocapsid, phosphoprotein, matrix, fusion protein and haemagglutinin genes were 1.5%, 1.5%, 1%, 0.75% and 0.5%, respectively, of the total cDNA library.

Animals↗

PubMed1999

Coadministration of interleukin 12 expression vector with antigen 2 cDNA enhances induction of protective immunity against Coccidioides immitis.

Interleukin 12 (IL-12) plays an important role in the induction of protective immunity against cancer and infectious diseases. In this study we asked whether IL-12 cDNA could increase the protective capacity of the antigen 2 (Ag2) gene vaccine in experimental coccidioidomycosis. Coimmunization of BALB/c mice with a single-chain IL-12 cDNA (p40-L-p35) and Ag2 cDNA, both subcloned into the pVR1012 plasmid, significantly enhanced protection against systemic challenge with 2,500 arthroconidia, as evidenced by a greater-than-1.3-log-unit reduction in the fungal load in the lungs and spleens compared to mice receiving the pVR1012 vector alone, Ag2 cDNA alone, or IL-12 cDNA alone. The enhanced protection was associated with increased gamma interferon secretion; production of immunoglobulin G2a (IgG2a), IgG2b, and IgG3 antibodies to Coccidioides immitis antigen; and the influx of CD4(+) and CD8(+) T cells in lungs and spleens. When challenged by the pulmonary route, mice covaccinated with Ag2 cDNA and IL-12 cDNA were not protected at the lung level but did show a significant reduction in the fungal load in their livers and spleens compared to mice vaccinated with Ag2 cDNA or IL-12 cDNA alone. These results suggest that IL-12 acts as a therapeutic adjuvant to enhance Ag2 cDNA-induced protective immunity against experimental coccidioidomycosis through the induction of Th1-associated immune responses.

Animals↗

PubMed1985

Bacteriophage lambda vector for transducing a cDNA clone library into mammalian cells.

We have developed a bacteriophage lambda vector (lambda NMT) that permits efficient transduction of mammalian cells with a cDNA clone library constructed with the pcD expression vector (H. Okayama and P. Berg, Mol. Cell. Biol. 3:280-289, 1983). The phage vector contains a bacterial gene (neo) fused to the simian virus 40 early-region promoter and RNA processing signals, providing a dominant-acting selectable marker for mammalian transformation. The phage DNA can accommodate pcD-cDNA recombinants with cDNA of up to about 9 kilobases without impairing the ability of the phage DNA to be packaged in vitro and propagated in vivo. Transfecting cells with the lambda NMT-pcD-cDNA recombinant phage yielded G418-resistant clones at high frequency (approximately 10(-2]. Cells that also acquired a particular cDNA segment could be detected among the G418-resistant transformants by a second selection or by a variety of screening protocols. Reconstitution experiments indicated that the vector could transduce 1 in 10(6) cells for a particular phenotype if the corresponding cDNA was present as 1 functional cDNA clone per 10(5) clones in the cDNA library. This expectation was confirmed by obtaining two hypoxanthine-guanine phosphoribosyltransferase (HPRT)-positive transductants after transfecting 10(7) HPRT-deficient mouse L cells with a simian virus 40-transformed human fibroblast cDNA library incorporated into the lambda NMT phage vector. These transductants contained the human HPRT cDNA sequences and expressed active human HPRT.

Animals↗

PubMed2005

Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles.

BACKGROUND: Gene expression studies on non-model organisms require open-end strategies for transcription profiling. Gel-based analysis of cDNA fragments allows to detect alterations in gene expression for genes which have neither been sequenced yet nor are available in cDNA libraries. Commonly used protocols for gel-based transcript profiling are cDNA differential display (DDRT-PCR) and cDNA-AFLP. Both methods have been used merely as qualitative gene discovery tools so far. RESULTS: We developed procedures for the conversion of cDNA Differential Display data into quantitative transcription profiles. Amplified cDNA fragments are separated on a DNA sequencer and detector signals are converted into virtual gel images suitable for semi-automatic analysis. Data processing consists of four steps: (i) cDNA bands in lanes corresponding to samples treated with the same primer combination are matched in order to identify fragments originating from the same transcript, (ii) intensity of bands is determined by densitometry, (iii) densitometric values are normalized, and (iv) intensity ratio is calculated for each pair of corresponding bands. Transcription profiles are represented by sets of intensity ratios (control vs. treatment) for cDNA fragments defined by primer combination and DNA mobility. We demonstrated the procedure by analyzing DDRT-PCR data on the effect of secondary metabolites of oilseed rape Brassica napus on the transcriptome of the pathogenic fungus Leptosphaeria maculans. CONCLUSION: We developed a data processing procedure for the quantitative analysis of amplified cDNA fragments separated by electrophoresis. The system utilizes common software and provides an open-end alternative to DNA microarray analysis of the transcriptome. It is expected to work equally well with DDRT-PCR and cDNA-AFLP data and be useful particularly in research on organisms for which microarray analysis is not available or economical.

Brassica napus↗

PubMed1994

Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper protein. Molecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein.

The cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.

Amino Acid Sequence↗

PubMed2004

[Construction and characterization of cDNA library for lung cancer cell line YTMLC-90 isolated from Gejiu, Yunnan province].

AIM: To construct the cDNA library for Yunnan Gejiu human lung cancer cell line YTMLC-90. METHODS: The total RNA was extracted from YTMLC-90 cells and the first-strand cDNA was synthesized by reverse transcription with a modified oligo(dT) primer (containing Sfi I B digestion site). Simultaneously, the SMART oligonucleotide (contained Sfi I A digestion site) was utilized as a template that the first-strand of cDNA could be extended out the 5' terminal of mRNA. The ds cDNA was amplified by LD-PCR (long-distance PCR) and then digested with Sfi I(IA & IB). After fractionation of cDNA through CHROMA SPIN column, the ds cDNA was cloned into lambdaTripIEx2 vector which was then packaged. RESULTS: The unamplified cDNA library for human lung cancer cells consisted of 1.01 x 10(9) pfu/L independent clones in which the recombinant rate was about 93.2%. The clone number in the amplified cDNA library reached 5.24 x 10(12) pfu/L and the length of inserted exogenous cDNA sequence was 750-3 000 bp. CONCLUSION: The constructed cDNA library for YTMLC-90 cells has an excellent quality, which lays a solid foundation for further screening and cloning novel tissue-specific genes of human lung cancer.

Carcinoma, Squamous Cell↗

PubMed2004

[Super SMART cDNA synthesis technology for amplifying small amount of total RNA of peripheral blood eosinophils from asthma patients].

OBJECTIVE: To amplify double-strand cDNA from small amount of total RNA of eosinophils from asthma patients by Super SMART cDNA synthesis technique. METHODS: The eosinophils were purified from the peripheral blood of asthma patients before and after treatment by Percoll gradient centrifugation, from which the total RNA was extracted using TRIzol kit. First-strand cDNA synthesis and double-strand cDNA amplification were performed using Super SMART cDNA synthesis technique. The quality of the obtained cDNA was evaluated by gradient cDNA electrophoresis, and the amplification efficiency determined by cDNA quantification. RESULT: From 20 ng total RNA, 7.155 microg and 6.568 microg of the tester and driver double- strand cDNAs respectively were obtained successfully, and the result of electrophoresis indicated high quality and purity of the cDNA acquired. CONCLUSION: Super SMART cDNA synthesis technique can effectively amplify high-quality double-strand DNA from a very small amount of total RNA, which may facilitate the exploration of the mechanism of asthma from the genetic level.

Asthma↗

PubMed2004

Preliminary study on the gene expression profiles of bone marrow mononuclear cells from patients with myelo-dysplastic syndrome by using cDNA microarray.

cDNA microarray, recently applied to analyze gene expression profile of cancers, was difficult to be utilized in myelodysplastic syndrome (MDS) for a special need of excessive mRNA hardly provided by ordinary bone marrow aspiration in MDS patients. The aim of this study was to investigate the feasibility of exploring the molecular events underlying MDS by using cDNA microarray and mixing mRNA from multiple patients. A commercially purchased BioStar H141 cDNA microarray containing 14,110 clones of cDNA or EST was employed to analyze the gene expression profile of bone marrow mononuclear cells from two cases of MDS. Equal amount of total RNA from each patient was mixed, reversely transcribed to cDNA and labeled with Cy5. Mixture of Cy5-labeled cDNA and Cy3-labeled cDNA from normal bone marrow cells was concomitantly hybridized to H141 microarray in duplicate. In H141 chips, 1,064 cDNAs were spotted at least twice targeting different fragments of a single gene cDNA. The results showed that among these 1,064 cDNA clones, the expression level of 625 (58.7%) and 630 (59.2%) ones was consistent within these two chips, respectively, 297 (27.9%) and 191 (18.0%) inconsistent, 21 (2.0%) and 11 (1.0%) in opposite. Among 411 duplicately spotted cDNAs with complete data, expression levels of 400 (97.3%) was consistent between two chips. 488 genes with known function were identified as differentially expressed in MDS, among which 101 genes were involved in hematopoiesis regulation, including those encoding transcription factors, cell cycle-regulating proteins, metabolism-relating enzymes, and adhesive molecules. It is concluded that cDNA microarray can be used for profiling gene expression of mixed MDS samples and replication shall be necessary for reducing the data bias caused by experimental operation.

Bone Marrow Cells↗

PubMed2005

[Screening of prothrombotic state-related genes from human hepatic cDNA library].

OBJECTIVE: To screen prothrombotic state-related cDNA sequences from human hepatic cDNA library. METHODS: The subtracted cDNA library for differentially expressed genes in rat liver of prothrombotic state (PTS) was constructed by suppression subtractive hybridization. Positive clones were identified by differential screening. The target DNA sequences of positive cDNA clones of differentially expressed genes from rat liver of PTS were amplified by PCR. The PCR products were used as probes to screen a human hepatic cDNA library. After the first, second and third screening, the transduction of a positive lambdaTripIEx lysate into E. coli strain BM25. 8 promoted the circularization of pTripIEx. The positive circularized plasmids were identified by double enzyme digestion. The cDNA fragments of the positive plasmids were sequenced and analyzed by bioinformatics (blastn). RESULTS: Four PTS-related cDNA sequences were identified from human hepatic cDNA library. For 3 of them, their products of expression were respectively fibrinogen, LFIRE1, and chromosome 10 open reading frame 104 mRNA. The fourth sequence had homology with carbamoyl-phosphate synthetase 1 gene. CONCLUSION: Four PTS-related cDNA sequences have been identified from human hepatic cDNA library.

Base Sequence↗

PubMed1985

Expression of polypeptide segments of the human complement component C3 in E. coli: genetic and immunological characterization of cDNA clones specific for the alpha-chain of C3.

The third component of complement C3 and its fragments have a central role in a variety of host defense mechanisms. The identification of functionally relevant C3 domains is important because of the marked functional versatility of the C3 molecule. Several human C3 cDNA clones from a human liver cDNA library were isolated and characterized. A bacterial expression vector system was used to express cDNA clones that were identified by an immunological screening procedure. The C3 cDNA clones produced in E. coli the hybrid proteins consisting of cro-beta-galactosidase and polypeptide segments of human C3, as revealed by Western blotting with antisera to human C3. The C3 moiety of the hybrid proteins had a m.w. of up to 46.000. Polyclonal antibodies against the C3 segments expressed by one of the C3 cDNA clones (ReC3-1) have been raised in mice and rabbit, and in addition, a monoclonal antibody was produced. The antisera and the monoclonal antibody reacted in Western blotting analysis selectively with the alpha-chain, but not the beta-chain of human C3. Restriction mapping of the different cDNA clones was performed, and revealed that the different clones were partially overlapping. The ReC3-1 cDNA clone included a 0.7 kb noncoding region at the 3' terminal end of the C3 cDNA. One of the restriction sites (Hind III) identified in the ReC3-1 cDNA clone was not present in the recently published sequence of human C3 cDNA. This difference in nucleotide sequence provides direct evidence for C3 polymorphism at the DNA level. The combination of immunologic procedures with recombinant DNA methodology should facilitate additional analysis of the structure-function relationship of the C3 molecule.

Animals↗

PubMed1988

Molecular cloning of cDNA corresponding to mRNA species whose steady state levels in the thyroid are enhanced by thyrotropin. Homology of one of these sequences with ferritin H.

A lambda gt11 cDNA library was constructed using poly(A)+ mRNA from thyrotropin (TSH)-stimulated Fisher rat thyroid (FRTL5) cells. The library was screened for nonthyroglobulin cDNA sequences by differential plaque filter hybridization using single-stranded cDNA probes synthesized from mRNA prepared from quiescent and TSH-stimulated FRTL5 cells. Thyroglobulin cDNA-containing recombinants in the library were avoided by prehybridizing the TSH probe to excess thyroglobulin cDNA. Of 48,000 clones screened, 60 were chosen as representing mRNA species whose abundance was increased in TSH-stimulated versus quiescent cultures. Southern blot analysis of 9 clones confirmed that the TSH-cDNA probe hybridized to a greater extent to the cDNA inserts than did the control probe. cDNA insert sizes varied between 0.3 kilobase (kb) and 1.0 kb. Northern slot blot analysis using as probes the cDNA of four of these clones (FC4, FC26, FC29, and FC43) demonstrated that TSH stimulation of FRTL5 cells increased the steady state levels of the respective mRNA species by 4-12-fold. For all 4 clones, increases in mRNA levels were apparent within approximately 1 h and were maximal after 14-18 h of TSH stimulation. Determination of the partial nucleotide sequence of these 4 clones confirmed that none was thyroglobulin, thyroid peroxidase, or any other gene previously reported to be stimulated by TSH. Three of the clones bore no homology to any known nucleotide sequence, but FC26 was 85% homologous with human ferritin H. Northern blot analysis using the FC26 cDNA insert as a probe confirmed hybridization to an mRNA species of 1 kb, the known size of ferritin H mRNA. In summary, using the technique of differential plaque filter hybridization, we have identified 4 new genes whose mRNA levels are increased by TSH stimulation of thyroid cells. One of these genes is homologous to human ferritin H.

Animals↗

PubMed1995

Cloning of rat lysosomal acid lipase cDNA and identification of the mutation in the rat model of Wolman's disease.

Lysosomal acid lipase (LAL) is a hydrolase essential for the intracellular degradation of cholesteryl esters and triglycerides. We previously reported a rat model of Wolman's disease (Wolman rat) that is deficient for LAL activity. In this study, we cloned rat LAL (RLAL) cDNA and investigated abnormal LAL gene expression in the Wolman rat. We cloned the RLAL gene from a cDNA library made from normal rat liver mRNA using the human LAL cDNA as a probe, subcloned the RLAL cDNA into pBlueScript vector, and sequenced it. Next, we constructed a cDNA library from a Wolman rat liver, and used the RLAL cDNA as a probe to isolate the Wolman RLAL cDNA for comparison. The normal RLAL cDNA contains 3150 bp including an 1194 bp open reading frame and three poly A signals at the 3' end. The deduced amino acid sequence contained 397 amino acids, showed 79.9% homology with human LAL, and had the same functional domains at the same sites as human LAL. Northern blot analysis revealed that the RLAL mRNA from normal rat was 3.2 kb in length, while the RLAL mRNA from Wolman rat was only 1.4 kb. Nucleotide sequence analysis showed that Wolman rat LAL cDNA had the same sequence as a RLAL cDNA from the 5'-untranslated region to nt 1101, followed by a 60 bp replacement from nt 1102 to nt 1161 with poly A signal and a 3' 1.8 kb deletion. The deduced amino acid sequence demonstrated the substitution of 367Ile to Asn, 368Pro to stop codon, and deletion of the C-terminal 29 amino acids. Genomic Southern blot analysis disclosed a large deletion at the 3' end of the gene. These results identify the molecular defect in the Wolman RLAL, and suggest that the C-terminus of RLAL is essential for the activity and/or stability of the enzyme.

Amino Acid Sequence↗

PubMed1997

Cloning and sequence analysis of human and bovine corneal antigen (CO-Ag) cDNA: identification of host-parasite protein calgranulin C.

PURPOSE: The primary structure of a cornea-associated antigen (CO-Ag) has been identified and has been implicated in the pathogenesis of Mooren's ulcer. The study designs were to isolate full-length clones encoding CO-Ag from a bovine and a human corneal cDNA library so that complete sequence analyses might further define the possible role of this protein in Mooren's ulcer. METHODS: DNA fragments of bovine and human CO-Ag were generated using unique oligonucleotide primers and reverse transcription polymerase chain reaction. These fragments were used as probes to obtain cDNA clones from a bovine and a human corneal cDNA libraries. The clones with the longest cDNA inserts were selected for sequence analyses. Human cDNA fragment was digested with Stu I and Hind III and cloned into a expression vector, pPROEXHT, at the same restriction enzyme sites. The plasmid was transformed into E. coli cells. Correct cloning and the full-length sequence of human CO-Ag were determined by sequencing the insert cDNA. RESULTS: The bovine cDNA insert sequence was 273 nucleotides in length for the entire mRNA coding region, 212 nucleotides in the 5' untranslated region, 83 nucleotides in the 3' untranslated region and a poly(A) tail. The DNA base sequence of this clone also contained a standard initiation codon, termination codon, and the polyadenylation signal. This cDNA predicts a protein which contains 91 amino acids with a molecular weight of 10,584 daltons. Plasmid expression vector, pPROEXHT-CO-Ag, was constructed that direct the synthesis of human CO-Ag in E. coli as fusion protein. Human CO-Ag fusion protein was purified to 90% pure with a yield of 17.2 mg per liter of the bacterial cell lysate. The nucleotide sequence of the CO-Ag cDNA insert was completely identical to human neutrophil calgranulin C. The deduced amino acid sequence was completely identical to a Ca(2+)-binding protein isolated on the surface of filarial nematodes. CONCLUSIONS: The isolation and analysis of cDNA clones containing the complete coding sequence of bovine and human CO-Ag proteins is reported. The proteins identified by deduced amino acid sequences demonstrate 100% sequence homology with human and bovine calgranulin C. Immune recognition of calgranulin C to a filarial nematode may lead to a hyperactive autoimmune response to CO-Ag in the cornea leading to a Mooren's ulcer.

Amino Acid Sequence↗

PubMed2003

Mice body weight gain is prevented after naked human leptin cDNA transfer into skeletal muscle by electroporation.

BACKGROUND: In this investigation, the feasibility of gene therapy for obesity by electroporational transfer of naked plasmid with leptin cDNA into skeletal muscle was tested. Both young and adult mice were studied. METHODS: Human leptin cDNA was attached to the human insulin precursor secretion signal peptide gene. The fused gene was then inserted into the mammalian expression vector pcDNA3.1(-) and transferred into skeletal muscle of normal female mice using electroporation. RESULTS: During the time of exogenic gene expression, daily food intake of leptin cDNA-treated mice was observed to be lower than the control. The body weight gain was prevented efficaciously regardless of if they were young or adult. At the 7th week after gene transfer, the body weight of both young and adult leptin cDNA-treated mice was about 20% lighter than the control. Although the body weight of pair fed controlled adult mice was close to the leptin cDNA-treated mice at the 8th week, they were always heavier than the leptin cDNA-treated mice before this time. The levels of retroperitoneal fats and serum TG of leptin cDNA-treated mice were markedly lower than that of the control. The relative serum hyperleptinemic level could last for about 2 months. The expression of leptin cDNA in muscle cells was also detected by RT-PCR. The levels of serum insulin and glucose of leptin cDNA-treated mice decreased slightly. Our data also showed that the hyperleptinmia resulted in uterus expansion in young mice, but not in the adults. CONCLUSION: The present study provides evidence of successful electroporation of naked plasmid DNA transfer for prevention or treatment of mice obesity.

Adipose Tissue↗

PubMed1994

In-tube cDNA cloning method using a biotinylated RNA probe.

We report a simple, practical method for isolating a particular cDNA from a single-stranded (ss) cDNA library in a tube without using a radioisotope. The method consists of three steps: (i) the capture of a target cDNA by a biotinylated RNA via intermolecular hybrid formation, (ii) the binding of the cDNA-RNA hybrid to an avidin-coated gel, (iii) the recovery of the target cDNA by degradation of the RNA under mild alkaline conditions. The effectiveness of the method was examined by isolating a clone carrying a metapyrocatechase gene from a model library. This model experiment achieved an enrichment of 4800-fold. This method was applied to cloning of a cDNA encoding interleukin 8 (IL-8) from a cDNA library prepared from phorbol myristate acetate-treated U937 cells. Using an RNA probe prepared from a truncated cDNA encoding IL-8, the corresponding full-length cDNA clones were successfully obtained.

Biotin↗

PubMed2000

Protein-ribosome-mRNA display: affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction.

An enzyme-ribosome-mRNA complex was specifically purified by binding to the immobilized enzyme substrate and the cDNA was cloned in a single-tube reaction by one-step reverse transcription-PCR. The ganglioside GM3, used by sialyltransferase II (ST-II) as a substrate, was coated on a 96-well microtiter plate and ST-II was in vitro transcribed and translated from a cDNA library. The isolation of an enzyme-specific protein-ribosome (PRIME) complex was achieved with as little as 0.1 ng ST-II-specific cDNA in 5 microg of a total plasmid preparation or with the cDNA prepared from sublibraries previously inoculated at a density of 2000 clones/culture well. The affinity purification of the PRIME complex was highly specific for GM3 and did not result in cDNA amplification when a different ganglioside (GM1) was used for coating of the microtiter plate. The amplified cDNA was used for cloning or a second round of ribosome display, providing a fast analysis of enzyme affinity to multiple substrates. PRIME display can be used for host-free cDNA cloning from mRNA or cDNA libraries and for binding site mapping of the in vitro translated protein. The use of a single-tube reaction in ligand-coated microtiter plates indicates the versatility of PRIME display for cDNA cloning by automated procedures.

Chromatography, Affinity↗

PubMed2000

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.

Astrocytoma↗