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At least 541 records · Page 30Linked to original sources
PubMed2002

[Cloning of differentially expressed cDNA sequences involved in malignant transformation induced by benzo(a)pyrene metabolite dihydroxyepoxy benzo pyrene].

OBJECTIVE: To clone differentially expressed cDNA sequences involved in malignant transformation induced by benzo(a)pyrene metabolite dihydroxyepoxy benzo pyrene (BPDE). METHOD: The malignant transformation of human bronchial epithelial cell line 16HBE induced by BPDE in vitro was used as a model for comparing gene expression between the transformed cells and controls. cDNA representational difference analysis (cDNA-RDA) was performed to isolate differentially expressed cDNA fragment in transformed cells. The cDNA fragments were ligated to pGEM-T vector and transformed into JM109 bacteria. The plasmid DNA were sequenced and compared with data in GenBank by BLASTN. RESULTS: Five cDNA sequences were found to be novel ones and were registered in dbest database, which assigned accession numbers in GenBank are BG354691, BG354692, BG354693, BG354694 and BG354695, respectively. Eight of the remaining cDNA sequences showed sequence homology to those previously reported such as ribosomal protein S23, MLN137, ACTN4, transforming growth factor and G protein gene. CONCLUSIONS: These 13 genes may be involved in BPDE-induced malignant transformation, but their biological characteristics and functions are left to further studies.

Benzopyrenes↗
PubMed2000

[Cloning and tissue expression pattern analysis of the human citrate synthase cDNA].

Tricarboxylic acid (TCA) cycle is an important way to generate ATP, which is widely distributed in the cells of animal, plant or microorganism. It catalyses the catabolism of sugar as well as protein and fat. Citrate synthase plays a key role in regulating TCA cycle and is responsible for catalysing the synthesis of citrate from oxaloacetate and acetyl CoA. Screening of genomic informatics was performed by using pig citrate synthase cDNA as a probe and a contig which is 1636 bp long and has highly homologous to the pig citrate synthase cDNA was obtained from selected ESTs with the ASSEMBLY program. According to the sequence of this contig, a pair of primers was designed and used to amplify cDNA libraries. A 1492 bp cDNA containing an open reading frame encoding 466 amino acids was cloned from human testis and skeletal muscle cDNA libraries. The deduced amino acid sequence of the cDNA showed 95%, 92% and 60.9% identity to pig, chicken and yeast citrate synthase respectively. Because the deduced amino acids sequence contains a highly conserved motif of citrate synthase from three different species, it is believed that this cDNA may be a transcript of human citrate synthase gene. Northern analysis showed that the human citrate synthase was expressed at high level in heart and muscle, at middle level in brain, kidney and pancreas tissues, not detectable in thymus and small intestine tissues, and at low level in other nine tested human tissues.

Amino Acid Sequence↗
PubMed2003

The isolation of cDNA clones from cucumber (Cucumis sativus L.) floral buds coming from plants differing in sex.

In this study, we found flower cDNA clones which may be connected with the development of flower sex in cucumber. Two pairs of nearly-isogenic lines: gynoecious GY3 (FFMMGG) versus hermaphrodite HGY3 (FFmmGG) and monoecious B10 (ffMMGG) versus gynoecious 2gg (ffMMgg) were used for clone isolation. To obtain differentially-expressed clones, we applied the differential screening method. 454 clones from GY3 and 478 from B10 cDNA libraries were isolated. The results of RFLP analysis with 56 cDNA clones showed no clones which cosegregated with sex in cucumber. The 28 cDNA B10 and 33 cDNA GY3 clones isolated using the differential screening method were sequenced. Some of them seem to may play a role in cell differentiation or flower development. Among the 61 identified clones, 14 show high homology to plant proteins, although of unknown function. 11 show high homology to known proteins, and the possible function of some of them is discussed. For 3 clones, no significant similarity was found. The 31 clones displayed high homology to plant cDNA in EST database. The patterns of expression of five differential cDNA clones, 35GY3, 216GY3, 47GY3, 100B10 and 157B10, were analyzed in cucumber flower buds using in situ RT-PCR. The most interesting clone is 35GY3, because of its possible role in the inhibition of the development of male specific elements in the female cucumber flower.

Amino Acid Sequence↗
PubMed2002

[Analysis of lecithin-cholesterol acyltransferase cDNA and protein sequence from tree shrew].

OBJECTIVE: To acquire cDNA sequence of lecithin-cholesterol acyltransferase (LCAT) from tree shrew and analyze the sequence structure. METHODS: The first strand cDNA was acquired by reverse transcription using mRNA from tree shrew liver as template. By the method of SMART RACE PCR, tree shrew LCAT cDNA was acquired and deduced its amino acids sequence. The sequence and structure of tree shrew LCAT cDNA and amino acid were analyzed and predicted by the molecular software. RESULTS: Tree shrew LCAT cDNA is composed of 1,340 bp, including 2 bp 5' untranslated region (5' UTR), 1,320 bp open reading frame (ORF) which encodes protein precursor of 440 amino acids (24 amino acids signal peptide and 416 amino acids mature peptide), and 18 bp 3' untranslated region (3'UTR). The stop codon is TAA and there is a poly (A) signal sequence AATAAA and a 25 bp poly (A) tail. Tree shrew LCAT cDNA sequence has been accepted by GenBank as a new gene, accession number AF272861 and its homology with human and baboon was 90% and 89%, respectively. CONCLUSION: The sequence of LCAT cDNA in tree shrew has high identity with that of human and other experimental animal species.

Amino Acid Sequence↗
PubMed2003

[Construction and significance of directional expression cDNA library from myeloid leukemia cell line U937].

To construct the cDNA expression library from human U937 cell, total RNA and purified mRNA in myeloid leukemia cell line U937 were extracted. The first and second strand of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with EcoR I adapters, and the end of EcoR I adapters was phosphorylated. Then the cDNAs were digested by Xho I, and the fragments smaller than 400 bp were removed by Sephacryl-S400 spin column, the fragments longer than 400 bp were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP expression vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the U937 cell line cDNA library consisting of 2.87 x 10(6) recombinant bacteriophages was constructed. The average size of exogenous insert in the recombinants was about 1.7 kb. It is concluded that the constructed cDNA library can be used to screen target clones.

Gene Library↗
PubMed2003

[Rapid construction and identification of full-length cDNA library of human glioma tissues].

OBJECTIVE: To explore a method for rapid construction of a full-length cDNA library of human glioma tissues using switching mechanism at 5' end of RNA transcript (SMART). METHODS: The total RNA was extracted from several samples of human glioma tissues and the mRNA was subsequently separated. Multiple mRNA samples were mixed to be used as the template for the first-strand cDNA synthesis. The CDS /3' PCR primer (containing Sfi IB site) was used in the first-strand reaction, and the SMART IV Oligo(dT) (containing Sfi A site) served as the short, extended template at the 5' end of the mRNA. With the above two primers, the primer-extension step generated full-length double-strand cDNA, which was digested by Sfi I restriction enzyme and ligated to the Sfi I A & B -digested lambdaTriplEx2 vector. The ligated vector was then packaged by lambda packaging extract for the final construction of the cDNA library. RESULTS: The unamplified human glioma cDNA library consisted of 2.4x10(6) independent clones with a recombination rate of 100%. The titer of the amplified cDNA library was 4.5x10(9) pfu/ml, and the average exogenous inserts of the recombinants was 1.2 kb in length. CONCLUSION: A high-quality full-length cDNA library of human gliomas was constructed successfully, which may facilitate further study of the screening and cloning of new tumor suppressor genes and tissue-specific genes of human glioma.

DNA, Complementary↗
PubMed1990

Evidence of introduction by molecular cloning of artificial inverted sequence at the 5' terminus of the sense strand of rat insulin-like growth factor-I cDNA.

A cDNA of insulin-like growth factor (IGF)-I mRNA was obtained from a rat liver cDNA library. This cDNA contained a 5' terminal sequence of 48 base pairs complementary to a sequence in the 3' terminal region (an inverted repeat sequence) except for one nucleotide insertion in the 3' region. Base sequence analysis of this cDNA, northern blot analysis of rat liver mRNA using a probe specific to 5' region of cDNA, base sequence studies of the 5' upstream region of a clone of the rat IGF-I gene, and some other studies suggested strongly that the inverted repeat sequence of the IGF-I cDNA used was introduced artificially during preparation of the cDNA library.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of a cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase.

Nitric oxide, which accounts for the biological activity of endothelium-derived relaxing factor (EDRF), is synthesized in endothelial cells from L-arginine by nitric oxide synthase (NOS). We report here the cloning and functional expression of a cDNA encoding human endothelial NOS. Oligonucleotides corresponding to amino acid sequences shared by cytochrome P450 reductase and the recently identified brain NOS were synthesized to amplify a partial cDNA encoding a bovine endothelial cell NOS-related protein. This partial cDNA was used to isolate a cDNA encoding a human vascular endothelial NOS. The translated human protein is 1294 amino acids long and shares 52% of its amino acid sequence with brain NOS. Using RNA blot hybridization, abundant endothelial NOS mRNA was detected in unstimulated human umbilical vein endothelial cells. To determine the functional activity of the endothelial protein, we ligated the cDNA into an expression vector and transfected it into NIH3T3 cells. Cells expressing this cDNA contained abundant NADPH diaphorase activity, a histochemical marker for NOS. In co-culture assays, nitric oxide production by transfected cells increased guanylate cyclase activity in reporter rat fetal lung fibroblasts. In addition, NOS-catalyzed conversion of arginine to citrulline in transfected cells was significantly increased by A23187, a calcium ionophore. Isolation of a cDNA encoding a calcium-regulated, constitutively expressed human endothelial NOS, capable of producing EDRF in blood vessels, will accelerate the characterization of the role of this enzyme in normal and abnormal endothelial regulation of vascular tone.

3T3 Cells↗
PubMed1992

Molecular cloning of AMP deaminase isoform L. Sequence and bacterial expression of human AMPD2 cDNA.

Human AMPD2 cDNA clones have been isolated from T-lymphoblast and placental lambda gt11 libraries utilizing a previously cloned rat partial AMPD2 cDNA as the probe. Alignment analysis of all cDNA clones indicates the presence of intervening sequences in several placental isolates. This has been confirmed by sequencing human AMPD2 genomic clones. Intervening sequences can be removed from the cDNA clones by restriction with endonucleases at unique sites within the proposed open reading frame. This results in a 3292-base pair cDNA proposed to contain the entire AMPD2 open reading frame, which would encode a 760-amino acid polypeptide with a predicted subunit molecular mass of 88.1 kDa. Nucleotide and predicted amino acid comparisons with the 264 base pairs of proposed coding sequences in the rat AMPD2 cDNA demonstrate 91% similarity and identity, respectively. A comparison of the predicted human AMPD1 and AMPD2 polypeptides demonstrates homology in their C-terminal domains. Included in this region is the conserved motif, SLSTDDP, proposed to be part of the catalytic site of all AMP deaminases. In contrast, the predicted N-terminal domains of the human AMPD1 and AMPD2 polypeptides are unique. When placed in a prokaryotic expression vector, the human AMPD2 cDNA expresses AMP deaminase activity which can be precipitated with polyclonal antisera specific for isoform L.

AMP Deaminase↗
PubMed2003

[Cloning and sequence analysis of a novel stage-specific cDNA from adult Trichinella spiralis].

OBJECTIVE: To clone a stage-specific novel cDNA from 5 day-old adult worm (AD5) of Trichinella spiralis. METHODS: The cDNA library of AD5 was screened by an AD5 stage-specific cDNA probe labeled with digoxigenin (DIG). The positive clones were sequenced and analysed. RESULTS: The positive clone contained a cDNA insert of 1,132 bp in length with a full length open reading frame (ORF) of 1,032 bp. The cDNA encoded a polypeptide of 343 amino acid residues(aa) with a molecular weight of 35.1 kDa and an isoelectric point (IP) of 4.8. InterProScan analysis showed that the 117-120 aa (SGYG) was a glycosaminoglycan attachment site, 27-86 aa was nematode cuticle collagen N-terminal domain and 153-228 aa was collagen repeat (G-x-y) domain. Signal PV2.0 analysis indicated that the region of 1-43 aa was a signal peptide. Blastn homology analysis in Genbank revealed that the cDNA had no obvious homology to any other known gene sequence. Blastn analysis revealed high homology to cuticle collagen with identities more than 40%. CONCLUSION: A novel AD5 stage-specific cDNA encoding a full length ORF was cloned and sequence analysis showed this gene encoded cuticle collagen of Trichinella spiralis.

Amino Acid Sequence↗
PubMed2003

[Cloning and sequence analysis of Eg95 cDNA from different stages of Echinococcus granulosus in Xinjiang].

OBJECTIVE: To study expression and sequence differences of Echinococcus granulosus 95(Eg95) antigen cDNA from different stages of protoscolex, oncosphere and adult worm of E. granulosus from Xinjiang Uighur Aut. Reg. METHODS: In accordance with the sequence of Eg95 antigen cDNA, the primers of Eg95 were designed. Eg95 antigen cDNAs were amplified by PCR from protoscolex, oncosphere and adult worm cDNA libraries of E. granulosus, respectively and were cloned into pUCm-T plasmid, and sequenced. The sequences were analyzed by DNAman and GenBank/BLAST biosoftware. RESULTS: PCR results showed that Eg95 antigen cDNA was amplified from three stages of E. granulosus cDNA libraries. Sequencing analysis indicated that the Eg95 cDNA length was 402 bp, same as the reported data in GenBank. CONCLUSION: The Eg95 antigen cDNA was expressed in the different life-cycle stages of E. granulosus in Xinjiang and there was no nucleic acid sequence difference of Eg95 antigen among the protoscolex, oncosphere and adult worm of E. granulosus.

Animals↗
PubMed2003

[Development of the cDNA chip for SARS virus and a primary study on the possible molecular mechanism of interferon alpha2b inhibiting the SARS virus replication].

BACKGROUND: To study the molecular mechanism of interferon alpha2b(IFNalpha2b) inhibiting the SARS virus replication. SARS-associated coronavirus (SARS virus) cDNA chip was developed and applied to detect the virus RNA transcription levels in the interferon-treated and untreated cell cultures, and the mechanism of anti-SARS virus activity of interferon alpha2b in cell culture system was explored. METHODS: SARS virus cDNA chip was prepared by comparing the published SARS virus genome sequence, and the cDNA chip was used to study the interferon alpha2b function during SARS virus replication. RESULTS: SARS virus cDNA chip was successfully prepared by using PCR method. The results showed that the cDNA chip could be used to detect the viral RNA transcription level. Interferon alpha2b could inhibit almost all the SARS virus gene transcription. An unknown gene at the position 28130-28426 bp, named as U gene, may play an important role during the viral replication. CONCLUSION: A SARS virus whole genome cDNA chip was established. It could be used to study the virus molecular biology and antiviral drug screening. The results also showed that interferon alpha2b could inhibit almost the whole virus gene transcription by using the cDNA chip.

Humans↗
PubMed2004

[Construction and analysis of subtractive cDNA library associated with multidrug resistance of acute leukemia].

The study was aimed to construct subtractive cDNA library associated with multidrug resistance (MDR) of acute leukemia for screening genes related to MDR in leukemia. The improved PCR-based subtractive hybridization was performed to clone differential genes between HL-60/VCR and HL-60 cell line. The mRNA of HL-60/VCR and HL-60 cell line were isolated. Then the mRNA of HL-60/VCR group was reversely transcribed into cDNA by Cap-Finder method, and the mRNA of HL-60 was reversely transcribed into cDNA by ordinary method to be marked by biotin for the hybridization next with HL-60/VCR cDNA. After hybridizing, filtrating through the sephacryl S-400 column, absorbing by the magnetic beads, and amplifying by PCR method, the fragments were cloned by T-A method and the cDNA library was constructed. Then the quality of cDNA library was identified by dot-blotting hybridization method. The results showed that after constriction, the library demonstrated its good quality. There was a high proportion of large fragments in this library. From small amount of samples a large amount of candidate fragments could be screened rapidly at once by dot-blotting hybridization. It is concluded that a differentially-expressed subtractive cDNA library in MDR of leukemia with high quality and larger fragments can be efficiently constructed by improving subtractive hybridization and selective PCR method.

Acute Disease↗
PubMed1992

Molecular cloning of the human cDNA for a stimulatory GDP/GTP exchange protein for c-Ki-ras p21 and smg p21.

We have previously purified smg GDP dissociation stimulator (GDS) from bovine brain and isolated its cDNA from a bovine brain cDNA library. smg GDS stimulates the GDP/GTP exchange reaction of a group of small GTP-binding proteins (G proteins), including at least c-Ki-ras p21, smg p21A, smg p21B, rhoA p21 and rhoB p21, by stimulating the dissociation of GDP from and the subsequent binding of GTP to each small G protein. In this study, we have isolated and sequenced the cDNA of smg GDS from a human brain cDNA library using the cloned bovine smg GDS cDNA. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,122. Human smg GDS shares 93% nucleotide and 96% amino acid sequence homologies with bovine smg GDS. The isolated cDNA is expressed in Escherichia coli, and the encoded protein shows the physical and functional properties similar to those of bovine smg GDS.

Amino Acid Sequence↗
PubMed2004

[Rapid construction of full-length MnSOD cDNA of chickens by one-step 3'RACE].

RACE (rapid amplification of cDNA ends) is a popular technique to rapidly obtain the full-length cDNA. After obtaining the 3' cDNA and 5' cDNA fragments with a overlapped region by 3' RACE and 5' RACE, the full-length cDNA could be generated by end-to-end PCR or subcloning. In this study, 3' RACE combined with touch-down PCR was successfully used for the rapid construction of full-length MnSOD cDNA of chickens. Compared with the conventional end-to-end PCR or subcloning, this method, called one-step 3' RACE, is fast, economical and highly specific. It especially fits the rapid construction of full-length cDNA by RACE method.

Animals↗
PubMed1992

Molecular cloning, characterization, and expression in Escherichia coli of a cDNA encoding mammalian choline kinase.

A rat liver lambda gt11 cDNA library was screened with antibody against rat liver choline kinase. One of the putative phage clones obtained was found to express choline kinase activity in infected Escherichia coli cells and used for the isolation of fully extended cDNA clones by hybridization. The obtained clones had identical overlapping nucleotide sequences and gave a composite cDNA sequence of 2540 bases. Poly(A)+ tails were found at three different sites of the cDNA sequence. Within the sequence there was a large open reading frame encoding 435 amino acids with a molecular mass of 49,743 Da. When this open reading frame was placed after the trc promoter and introduced into E. coli cells, fully active choline kinase was produced. This enzyme was partially purified using a published choline kinase purification method and was found to mediate the phosphorylation of choline, N,N-dimethylethanolamine,N-monomethylethanolamine, and ethanolamine. Similar to the case with choline kinases from various sources, Km decreased as the number of N-methyl groups increased. The deduced amino acid sequence significantly resembled the yeast choline kinase sequence. It also showed local sequence similarity to protein kinases and some bacterial phosphotransferases. Northern blot analysis revealed that the cDNA probe hybridized to 2.8- and 1.8-kilobase RNA in various rat tissues. The sizes of the transcripts were fairly consistent with that of cDNA. The order of mRNA abundance was testis, brain, lung, kidney, and liver, but did not coincide with the order of the activity levels in these tissues. These results show that the cloned cDNA encodes one of the choline kinase isoforms present in mammalian tissues.

Amino Acid Sequence↗
PubMedDate not supplied

[Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages].

Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.

Base Sequence↗
PubMed2005

[Construction of eukaryotic expression vector of mouse alpha-fetoprotein cDNA and its expression in dendritic cells and in vitro antitumor effect on hepatoma].

BACKGROUND & OBJECTIVE: Studying the mechanism of why the abnormal protein coded by mutant gene of hepatoma carcinoma cell possesses potential antigenicity, but can not form effective immunogen, searching for specific antigen of hepatoma, and developing DNA vaccine for hepatoma treatment will have active impact on the immunotherapy of hepatoma. This study was to construct fluorescent eukaryotic expression vector of mouse alpha fetoprotein (AFP) cDNA fragment with or without the secretory signal peptide (termed AFP1 cDNA and AFP2 cDNA), and observe its expression in mouse dendritic cells (DCs) and in vitro antitumor effect on hepatoma. METHODS: AFP1 cDNA and AFP2 cDNA were amplified from mouse hepatoma cell line H22 by reverse transcription-polymerase chain reaction (RT-PCR), and cloned into eukaryotic expression vector PEGF-N3 to form the recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2. The mouse bone marrow cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to form mature DCs. The recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2 were transfected into DCs. The DCs vaccine were co-cultured with isogenous mouse spleen lymphocytes. The expression of gamma-interferon (IFN-gamma) in the spleen lymphocytes stimulated by AFP1/DCs or AFP2/DCs was measured by ELISA assay; the specific killing activity of cytotoxic T lymphocytes (CTLs) was evaluated by 51Cr release assay. RESULTS: Sequencing analysis verified that AFP1 cDNA and AFP2 cDNA were correctly amplified from H22 cells. The recombinant PEGF-N3/AFP1 and PEGF-N3/AFP2 were stably expressed in mouse DCs. AFP2/DCs obviously stimulated proliferation of T lymphocytes; the stimulation index (SI) was significantly higher in AFP2/DCs group than in AFP1/DCs group, empty vector group, and blank control group (5.12 +/- 1.46 vs. 1.41 +/- 0.65, 1.42 +/- 0.7, and 1.41 +/- 0.55, P < 0.01); the specific killing activity of CTLs was stronger in AFP2/DCs group than in empty vector group [(88.15 +/- 16.4)% vs. (12.72 +/- 5.45)%]. CONCLUSION: The recombinant expression vectors PEGF-N3/AFP1 and PEGF-N3/AFP2 with or without signal peptide were constructed successfully. AFP2/DCs can specifically activate T lymphocytes in vitro and effectively induce antitumor immune response.

Animals↗