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PubMed1993

Expression cloning of a cDNA encoding a novel murine B cell activation marker. Homology to human CD38.

A rat mAb (NIM-R5) has recently been prepared against a novel murine B cell activation marker. We report here isolation of a cDNA (1-19) encoding the B cell-derived protein recognized by NIM-R5 antibody. This cDNA contains an open reading frame that encodes a polypeptide of 304 amino acids with a predicted molecular weight of 34,500. The existence of a 22-amino acid hydrophobic region located 23 amino acids from the amino terminal of the deduced protein, together with four potential N-linked glycosylation sites, characterize the deduced protein encoded by I-19 cDNA as a typical type II transmembrane glycoprotein. Although I-19 cDNA appears to encode a novel murine protein, its nucleotide sequence and deduced amino acid sequence show approximately 70% homology to the previously reported sequence of human CD38, suggesting that I-19 cDNA encodes either the mouse homologue of CD38 or a closely related protein. Northern blot analysis of the expression of this cDNA product in a variety of cell types, together with immunoprecipitation of the recombinant protein expressed in BaF3 cells, indicated that I-19 cDNA encodes not only the epitope recognized by NIM-R5 but also a protein that is indistinguishable biochemically and in terms of distribution from the murine B cell activation marker recognized by NIM-R5 antibody. Chromosomal mapping studies have localized this locus to the proximal region of mouse chromosome 5. We anticipate that the availability of probes for the murine B cell activation marker recognized by NIM-R5, and the recombinant protein itself, will greatly aid efforts to define the role of this molecule in murine B cell development.

ADP-ribosyl Cyclase↗
PubMed1993

A novel form of cytochrome P-450 family 4 in human polymorphonuclear leukocytes. cDNA cloning and expression of leukotriene B4 omega-hydroxylase.

Isolation of cDNA clones for human leukotriene B4 (LTB4) omega-hydroxylase clearly demonstrates that the hydroxylase is a member of the cytochrome P-450 (CYP) superfamily. cDNA clones isolated from a human leukocyte cDNA library with CYP4A4 cDNA as a probe encode a protein of 520 amino acids with a molecular weight of 59,805. The deduced amino acid sequence contains an invariant cysteine in the conserved heme-binding domain near the C terminus, characteristic of the P-450 superfamily. The microsomes from yeast cells transfected with an expression vector pAAH5 carrying isolated cDNA catalyzed the omega-hydroxylation of LTB4 with a Km value of 0.71 microM, and its activity was significantly inhibited by carbon monoxide and by antisera against CYP4A4, consistent with the properties previously reported with LTB4 omega-hydroxylase in human polymorphonuclear leukocytes. The amino acid sequence of LTB4 omega-hydroxylase (P-450LTB omega) shows 31-44% similarity to those of CYP4A, CYP4B, and CYP4C, whereas less than 25% similarity was observed with any of the other P-450 families. According to the systematic classification of the P-450 superfamily, P-450LTB omega is classified into the CYP4 family but does not belong to any of the known CYP4 subfamilies. This P-450 composes a new subfamily of CYP4. RNA blot analysis indicated that mRNA hybridized to the cDNA was expressed in the polymorphonuclear leukocytes as well as leukocytes from four individuals. Isolation of the cDNA opens the way to investigate the physiological role and to regulation of the omega-hydroxylase in the inflammation process.

Amino Acid Sequence↗
PubMed1997

AAC-11, a novel cDNA that inhibits apoptosis after growth factor withdrawal.

Many growth factors and cytokines act as cellular survival factors by preventing programmed cell death (apoptosis). However, the specific genes and corresponding proteins that mediate survival are poorly defined. To identify potential survival genes, a cDNA library was prepared from murine fibroblasts and screened by a functional expression cloning approach. A 1023-bp cDNA, AAC-11, was identified that encodes a protein of approximately 25 kDa. The AAC-11 gene shows strong species conservation and is ubiquitously expressed in embryonic and adult tissues with multiple transcripts, as well as in various human tumor cell lines. The predicted protein contains a leucine zipper domain but lacks a DNA-binding domain. BALB/c3T3 fibroblasts that were stably transfected with AAC-11 cDNA were viable in serum-free medium for up to 12 weeks. The protective action of AAC-11 was abolished by mutation of leucines to arginines within the leucine zipper domain. We also isolated a longer AAC-11 cDNA that codes for up to an additional 290 amino-terminal amino acids but did not protect against apoptosis. The cDNA for human AAC-11 was identified and exhibits strong homology with the murine species and retains the leucine zipper domain. Western immunoblots of BALB/c3T3 cells using rabbit anti-AAC-11 polyclonal serum revealed a major native 55-kDa AAC-11 protein and a minor 25-kDa protein corresponding to the long and short forms of AAC-11 cDNA, respectively. In summary, we report a cDNA whose expression supports cell viability after withdrawal of growth factors. The corresponding native protein may function as a novel inhibitor of apoptosis.

Alternative Splicing↗
PubMed1998

Isolation and partial characterization of a cDNA encoding a rabbit liver carboxylesterase that activates the prodrug irinotecan (CPT-11).

We have isolated a cDNA encoding a rabbit carboxylesterase (CE; EC 3.1.1.1) that converts the camptothecin-derived prodrug irinotecan (CPT-11) to the potent topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin. NH2-terminal amino acid sequencing of a purified rabbit CE allowed the design of redundant oligonucleotides to perform PCR from rabbit liver cDNA. DNA sequencing of the PCR product confirmed the identity of the clone, and after both 5' and 3' rapid amplification of cDNA ends, oligonucleotide primers were designed to amplify the entire cDNA. The 1698-bp open reading frame encoded a 565-amino acid protein containing the characteristic CE B-1 and B-2 motifs, a hydrophobic NH2-terminal leader sequence, and the COOH-terminal residues HIEL that are thought to be responsible for protein localization in the endoplasmic reticulum. Transient expression of the cDNA in COS-7 cells resulted in CE activity in cell extracts and increased the sensitivity of cells to CPT-11. Additionally, stable expression of the rabbit liver CE cDNA in the human glioma U-373 MG cell line resulted in a 56-fold decrease in the IC50 value for CPT-11, whereas the expression of a human alveolar macrophage cDNA encoding a highly homologous CE produced no change in drug sensitivity.

Amino Acid Sequence↗
PubMed1998

Cloning of a cDNA encoding a putative molt-inhibiting hormone from the eyestalk of the sand shrimp Metapenaeus ensis.

Degenerate primers were designed from the amino acid sequence of the neuropeptide Pej-SGP-IV of the shrimp Penaeus japonicus. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using eyestalk complementary DNA of the sand shrimp Metapenaeus ensis. A partial cDNA that codes for a protein homologous to the neuropeptide Pej-SG-IV was cloned. The partial cDNA was used as a probe to screen the eyestalk cDNA library. Several cDNA clones with nucleotide sequence identical to the partial cDNA were isolated. The largest cDNA is 957 bp with an open reading frame consisting of a coding sequence 315 bp in length. The deduced amino acid of the neuropeptide consists of 77 amino acids and is preceded by a signal peptide of 28 amino acids. Because the deduced amino acid sequence of the shrimp cDNA is highly homologous to the Pej-SGP-IV of P. japonicus (which is molt inhibiting) and to other crustaceans' molt-inhibiting hormones (MIHs), the shrimp neuropeptide is tentatively called MeMIH. Northern blot analysis and RT-PCR showed that MeMIH is expressed in the postmolt, intermolt, and premolt stages of the shrimp eyestalks and the brain. Moreover, RNA message can also be detected in the nervous tissues of newly developed larvae. MeMIH is, however, not found in the muscle, swimming leg, and hepatopancreas. Results from genomic Southern blot analysis and amplification of the shrimp genomic DNA by polymerase chain reaction (PCR) suggest that a single copy of the MIH gene is present in the genome. The structural organization of the gene for the shrimp putative MIH is similar to that of the crab Charybdis feriatus.

Amino Acid Sequence↗
PubMed1998

Sequence and structural analysis of feline interleukin-5 cDNA.

OBJECTIVE: To clone and characterize the cDNA encoding feline interleukin-5 (IL-5) cDNA and the 170 basepairs (bp) of the 5' flanking region of the feline IL-5 gene. SAMPLE POPULATION: Blood mononuclear cells from a healthy cat. PROCEDURES: Cells were cultured, stimulated for 48 hours with concanavalin A, and harvested for RNA and DNA isolation. Recovered RNA was used in northern blot and reverse transcription-polymerase chain reaction analyses. Resulting cDNA was used for rapid amplification of 3' cDNA ends, dideoxy chain termination sequencing, and primer extension analysis. RESULTS: Full length cDNA was 838 bp, including a 402-bp open reading frame that encoded a precursor protein of 134 amino acids including a putative peptide signal of 19 residues. Homologies of the nucleotide and derived protein sequences between feline and human IL-5 cDNA were 72 and 71%, respectively. There also was homology between the human and predicted feline cytokines at amino acid positions that are critical for IL-5 receptor binding and signal transduction. The 5' flanking region of the feline gene was homologous to corresponding regions of the human (88%) and murine (72%) genes, and included putative transcriptional regulatory elements. CONCLUSIONS AND CLINICAL RELEVANCE: Identification of feline IL-5 cDNA is an important step toward a detailed, fully comprehensive characterization of the mechanisms that may be operative in the pathogenesis of eosinophilic disorders in cats. The striking homology between the human and feline IL-5 genes suggests that cats can be used as animal models for human diseases characterized by eosinophil infiltration of tissues.

Amino Acid Sequence↗
PubMed1999

Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR.

Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a approximately 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P<0.001, n=4), and a approximately 4-fold increase in copy number with a specific hexamer (P<0.001, n=4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.

Base Sequence↗
PubMed2004

SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones.

BACKGROUND: cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library. RESULTS: We describe a method to construct SMART full-length enriched cDNA libraries with large insert sizes. Sub-libraries were generated from size-fractionated cDNA with an average insert size of up to seven kb. The percentage of full-length clones was calculated for different size ranges from BLAST results of over 12,000 5'ESTs. CONCLUSIONS: The presented technique is suitable to generate full-length enriched cDNA libraries with large average insert sizes in a straightforward and robust way. The representation of full-coding clones is high also for large cDNAs (70%, 4-10 kb), when high-quality starting mRNA is used.

Cell Line, Tumor↗
PubMed1999

In vivo construction of cDNA libraries for use in the yeast two-hybrid system.

We describe a simple and efficient one-step method to make cDNA libraries using homologous recombination in yeast. cDNA from any source, together with a linear vector, is used to transform yeast. Through homologous recombination and gap repair, the cDNA is unidirectionally incorporated into the yeast expression vector in vivo. The cDNA-encoded proteins can then be screened for potential protein-protein interactions with a bait already present in the yeast. This method allows rapid construction and screening of cDNA libraries, even from extremely small amounts of mRNA, and can replace the use of conventional cDNA libraries.

Cloning, Molecular↗
PubMed2004

Protection against MUC1 expressing mouse tumours by intra-muscular injection of MUC1 cDNA requires functional CD8+ and CD4+ T cells but does not require the MUC1 tandem repeat domain.

Using a C57Bl/6 mouse model system, where intramuscular (i.m.) injection of full length (FL) MUC1 cDNA protects against subsequent challenge with MUC1-expressing syngeneic tumour cells, we have investigated the importance of the tandem repeat (TR) domain in the induction of T cell-dependent tumour rejection. A MUC1 construct engineered to remove the TR domain (MUC1 0TR) was found to be as effective as the full length MUC1 cDNA in inhibiting the growth of RMA MUC1 cells in C57Bl/6 mice. Protection by i.m. injection of either the FL-MUC1 cDNA or the MUC1 0TR construct depended on the presence of functional CD4+ and CD8+ T cells. Specific CD8+ T cell responses, however, could not be detected in vitro using mouse spleen cells taken after only cDNA injection, but only after challenge in vivo with MUC1-expressing tumour cells. To attempt to enhance the responses of CD4+ T cells, a cDNA construct was developed, where the extracellular domain of MUC1 was fused to the transmembrane and cytoplasmic domain of Lamp1 (MUC1/Lamp1). This construct was equally effective in inducing tumour rejection but did not induce MUC1-specific CTL in mice before challenge with MUC1-expressing tumour cells. Our results indicate that, in this model, T cell responses necessary for protection against MUC1-expressing tumours that are induced by IM injection of MUC1 cDNA are independent of the tandem repeat domain as well as the transmembrane and cytoplasmic domains. A low level of protection was seen with all constructs in BALB/c mice, which show a defect in Th1 responses. C57Bl/6xBALB/c hybrids were, however, well protected against both H2(d) and H2(b) expressing tumour challenge, emphasizing the importance of the host background.

Animals↗
PubMed1984

The glyceraldehyde 3 phosphate dehydrogenase gene family: structure of a human cDNA and of an X chromosome linked pseudogene; amazing complexity of the gene family in mouse.

In an experiment designed to find sequences common to a skeletal muscle cDNA library and an X chromosome specific library, we have isolated cDNA clones corresponding to glyceraldehyde 3 phosphate dehydrogenase (GAPD), (whose gene is assigned to chromosome 12), and a DNA fragment from the X chromosome short arm which contains an intron-less GAPD pseudogene. A 1210-bp cDNA sequence has been established which covers all of the protein-coding region, most of the 5' non-coding region and part of the 3' non-coding region. It corresponds to the major (and possibly unique) GAPD mRNA present in skeletal muscle. Unexpectedly, the amino acid sequence derived from the cDNA clones differs at 10% of the residues from that established for the human protein purified from skeletal muscle. The X-linked pseudogene has been localised in the p22-p11 region of the human X chromosome. It has the structure of a complete retrotranscript of a processed mRNA, including the poly(A) tail and is 96% homologous to the cDNA sequence. The pseudogene is flanked by a 15-bp direct repeat, and an Alu-like sequence is found in the 3'-flanking region. About 25 GAPD sequences are found in the human genome, 12 of which have high homology to the cDNA probe. A similar complexity is found in hamster. In contrast, the mouse genome contains an amazing number of GAPD related fragments (at least 200). The hybridization pattern suggests that this multiplicity has been generated by two different mechanisms: first the generation of approximately 40 different sequences, which were subsequently amplified (probably by tandem duplication).

Animals↗
PubMed1998

Transduction of recombinant human erythropoietin receptor cDNA into daughter progenitors derived from single CD34(3+) cord blood cells changes the differentiation profile of daughter progenitors.

In this study, we tested the capacity to change the differentiation profile of progenitor cells by retroviral-mediated transduction of EpoR cDNA into one of the paired daughter cells derived from single CD34(3+) CB cells. Our results show that for the non-viral-treated daughter cells, the majority (99.6%) formed the same colony type. However, with cells transduced with viral vectors, 7.1% of the daughter cells transduced with the EpoR cDNA formed either a burst forming unit-erythroid (BFU-E) or a colony-forming unit-granulocyte, macrophage, erythroid, megakaryocyte (CFU-GEMM) colony compared to the other daughter cell transduced with viral supernatant lacking EpoR cDNA, which formed either a colony-forming unit granulocyte-macrophage (CFU-GM) or a high proliferative potential-colony forming cell (HPP-CFC) colony. Expression of the transduced EpoR cDNA was confirmed in individual colonies by RT-PCR analysis. These results substantiate in a more rigorous fashion our previous results that it is possible to change the Epo-responsive differentiation profile of progenitor cells by transduction into these cells of an EpoR cDNA and this change was apparent only in daughter cells derived from single CD34(3+) kit+ cells transduced with EpoR cDNA.

Antigens, CD↗
PubMed1994

Comparison of quantitative cDNA-PCR with the branched DNA hybridization assay for monitoring plasma hepatitis C virus RNA levels in haemophilia patients participating in a controlled interferon trial.

The branched DNA (bDNA) assay was compared with a semi-quantitative cDNA-polymerase chain reaction (cDNA-PCR) assay for monitoring HCV RNA levels in plasma in 17 haemophilia patients participating in a controlled alpha-interferon trial. Good correlation between the HCV RNA levels as detected by the two assays was observed, with a correlation co-efficient of 0.83 (P < 0.0001) and 0.90 (P < 0.0001) at week 0 and 24, respectively. Hepatitis C virus RNA (HCV RNA) levels could be assessed with the bDNA assay in 14/17 (82 percent) HCV cDNA-PCR positive pretreatment samples. The bDNA assay apparently failed to detect low viral titres. Interferon treated patients (n = 11) showed either a complete response, being a large reduction in HCV RNA level to below the detection limit of the HCV cDNA-PCR assay (6/11) or no significant reduction in HCV RNA level (5/11). A "partial" virological response was not observed. The changes in HCV RNA plasma levels in non-responders during interferon (IFN) treatment were similar to the (small) natural fluctuations in viral load observed in controls (untreated patients). Although the bDNA assay was not as sensitive as cDNA-PCR, given its user friendliness and quantitative results, it is concluded that it is a useful test for monitoring HCV RNA levels in patients treated with interferon. However, patients who are non-reactive in the bDNA assay have to be retested by cDNA-PCR because low viral titres are not detected by the bDNA assay.

Alanine Transaminase↗
PubMed2001

Cloning and sequencing of cDNA encoding for a novel human testis-specific contraceptive vaccinogen: role in immunocontraception.

Sperm-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific antigen. The 32P-labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -cDNA ends (5'-RACE) procedure. The full-length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of approximately 64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV antigen has a role in human sperm function and may find clinical applications in the contraceptive vaccine development and in the specific diagnosis and treatment of male infertility.

Adult↗
PubMed2006

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression.

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.

Amino Acid Sequence↗
PubMed2001

Transfection of MCF-7 carcinoma cells with human integrin alpha7 cDNA promotes adhesion to laminin.

The laminin-binding alpha7beta1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin alpha7 subunit contributes to a form of muscular dystrophy in integrin alpha7 null mice, whereas specific mutations in the alpha7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin alpha7 in human-related muscular disorders, we cloned alpha7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin alpha7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human alpha7X2B alternative splice form. Expression of human alpha7 was further confirmed by transfection of chimeric human/mouse alpha7 cDNA constructs. To demonstrate the functionality of expressed human alpha7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human alpha7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human alpha7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human alpha7 cDNA. These results show for the first time the exogenous expression of functional full-length human alpha7 cDNA, as well as the development of monoclonal antibodies against the human alpha7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving alpha7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.

3T3 Cells↗
PubMed1993

DNA-DNA subtractive cDNA cloning using oligo(dT)30-Latex and PCR: identification of cellular genes which are overexpressed in senescent human diploid fibroblasts.

We previously reported an efficient method for subtractive cDNA cloning using oligo(dT)30-Latex and polymerase chain reaction (PCR) (E. Hara et al., Nucleic Acids Res. 19, 7097-7104, 1991). The subtraction was performed by hybridization between mRNA of cell type B and the cDNA made from mRNA of cell type A using an oligo(dT)30 primer covalently linked to Latex particles in an Eppendorf tube. The mRNA common to both types of cells could be removed by a brief centrifugation. In the present paper, the method was improved by using the sense strand DNA instead of mRNA for hybridization to cDNA covalently linked to the particles to minimize mRNA degradation and by optimizing the hybridization condition. The sense strand DNA was made from cDNA-oligo(dT)30-Latex by asymmetric PCR. Using the improved method, a subtractive cDNA library with longer cDNA inserts was successfully constructed with higher probability than the original method.

Animals↗
PubMed2001

A strategy to identify differentially expressed genes using representational difference analysis and cDNA arrays.

Representational difference analysis (RDA) combined with cDNA arrays is an effective approach to identify differentially expressed genes. To identify differentially expressed genes in c-Myc transgenic mouse liver, we compared the virtues of probing commercially available cDNA arrays with either radiolabeled cDNA pools or radiolabeled difference products (DP2) derived from RDA using c-Myc transgenic and normal mouse liver. Probing commercial and custom arrays with DP2 products led to the identification of transcripts of low abundance that were missed when the arrays were initially probed with PCR-amplified cDNA pools. Although DP2 probes also detected abundant transcripts that are highly differentially expressed, they failed to identify abundant transcripts with low differential expression that were detected with cDNA pools. The combined use of radiolabeled cDNA and DP2 products to probe arrays allows a more comprehensive identification of differentially expressed transcripts that are abundant or rare. Our method has the additional benefit of eliminating false-positive transcripts that lack true differential expression and frequently contaminate DP2 pools. Using this method we identified 16 differentially expressed genes in c-Myc transgenic liver, one of which is novel.

Animals↗