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At least 883 records · Page 49Linked to original sources
PubMed2001

[Cloning and sequencing of prolactin gene cDNA in three chicken breeds].

The total RNA was extracted from the pituitary of two Chinese native chicken breeds, Yuehuang and Taihe Silkies, and one layer Isa, using RNeasy Kit (QIAGEN, Germany). The total RNA was used to synthesize a specific fragment with RT-PCR, in which the primers were designed based on the sequence of broiler prolactin gene cDNA. The specific fragment was ligated to a linear plasmid pBSSK and cloned with XL1-Blue. The sequencing of prolactin cDNA was carried out with ABI PRISH 377XL DNA Sequencer after cloning. The cDNA sequences and deduced amino acid sequences of prolactin gene of two Chinese native chicken breeds and one layer were compared with that of broiler, dwarf chicken and turkey. There was 93.97%-99.89% cDNA sequence homology among Chinese native, layer, broiler and dwarf chickens, in which there was the highest (99.87%) between those of Taihe and dwarf chickens. There was 98.25%-100% deduced amino acid sequence homology among Chinese native, layer, broiler and dwarf chickens, in which there was the highest (100%) between those of Taihe Silkies and dwarf chickens. It was found that Yuehuang and Taihe Silkies had the same signal peptide cleavage site Leu-Pro-Ile-Cys among amino acids sequence deduced from pre-prolactin cDNA as broiler, dwarf and turkey, while layer Isa had a different cleavage site Pro-Pro-Ile-Cys. Such difference might cause a different translation processing of pre-prolactin, which could make layer Isa non-broody. There were different amino acids in the positions 71, 141, 150 and 175 of deduced prolactin amino acid sequences among Yuehuang, Taihe Silkies, layer, broiler and dwarf chickens. There was a heparin-binding site in positions 175-181 (L-R-R-D-S-H-K) among prolactin amino acid sequences of broiler and Taihe Silkies.

Amino Acid Sequence↗
PubMed1999

[Cloning and partially sequencing of mouse dentin sialophosphoprotein encoding mature protein].

OBJECTIVE: Cloning and partially sequencing of mouse dentin sialophosphoprotein (DSPP) encoding mature protein. METHODS: Total RNA was extracted from the tooth germs of newborn mouse by acid guanidinium thiocyanata-phenol-chloroform method, the desired DNA product was obtained from the total RNA by RT-PCR with the primers including Oligo(dt) and two gene specific primers. The segment (about 3 Kbp) was inserted into pBluescript vector and the interesting plasmid was transformed into E. Coli host strain XL1-Blue. The double-stranded DNA of the positive clone was analyzed by restriction endonuclease mapping and DNA sequencing. RESULTS: The restriction endonuclease map and sequence of mouse DSPP encoding mature protein were consistent with those of the published. CONCLUSION: The mouse DSPP cDNA encoding mature protein was obtained for further study.

Animals↗
PubMed1999

Cloning and Expression of a Novel Mutated Osteoprogerin/Osteoclastogenesis Inhibitory Factor Gene.

Total RNA was isolated from normal Chinese human liver cell line L02. A mutated osteoprotegerin/osteoclastogenesis Inhibitory Factor(OPG/OCIF) cDNA was amplified by RT-PCR using the total RNA as template and was inserted into pBS-sk plasmid. Sequence analysis showed that the OPG/OCIF cDNA from L02 cells was a mutated OPG/OCIF gene which had a nonsense mutation(Ochre) at the codon of Gln(394). The OPG/OCIF isoform was 8 amino acid residues less at the C terminal than the OPG/OCIF reported. The 3'fragment of OPG/OCIF gene from genomic DNA of cell line 293(ATCC CRL 1573) was also cloned. Sequence analysis indicated that the sequence of genomic DNA was the same as that of cDNA. The mutated OPG/OCIF gene was inserted into yeast expression plasmid pPIC3.5K, and the recombinant plasmid was used to transform Pichia pastoris GS115. SDS-PAGE analysis revealed that the human OPG/OCIF isoform was highly expressed and accumulated up to over 30% of soluble protein of yeast after the induction by methanol for 3 to 5 days. The longer the transformant was induced, the higher the ratio of glycosylated protein was.

Journal Article↗
PubMed2004

[Cloning of human amelogenin gene encoding mature peptide].

PURPOSE: The purpose of this study was to clone human amelogenin gene encoding mature protein, which provides a basis for expressing the recombinant human amelogenin in Escherichia coli. in the future. METHODS: In this study, total RNA was extracted from the dental germ of a legally aborted embryo by Trizol. Using RT-PCR technique we obtained synthesis of cDNA from the total RNA, and the desired DNA products were conducted with PCR from cDNA. The segment was inserted into expression vector PQE30 and the interesting plasmid was transformed into Escherichia coli. host DH5alpha. The double-stranded DNA of positive clone was analyzed by PCR, restriction endonuclease mapping and DNA sequence analysis. RESULTS: The sequence analysis of recombinant plasmid showed that the human amelogenin encoding mature protein was inserted into vector PQE30 accurately. CONCLUSIONS: We conducted human amelogenin encoding mature protein from dental germ of a legally aborted embryo and got the recombinant plasmid which may express amelogenin gene for further research.

Amelogenin↗
PubMed1996

Differential expression of psoriasin messenger RNA between in situ and invasive human breast carcinoma.

The mRNA encoding the calcium-binding protein psoriasin was identified as being more highly expressed in the in situ versus the invasive component of the same breast tumor. Reverse transcription-PCR analysis of total RNA extracted from three independent cases of ductal carcinoma in situ of the comedo type and three cases of high-grade invasive ductal carcinoma revealed a detectable level of psoriasin mRNA expression in the in situ lesions only. Similar analysis performed on total RNA extracted from frozen sections of 32 independent breast samples, representing a continuum from normal to invasive tumor, confirmed high psoriasin expression in ductal carcinoma in situ of the comedo type only. The possible functional role of the psoriasin protein in breast tumor cells remains to be determined.

Breast Neoplasms↗
PubMed1998

Expression of placental leptin and leptin receptor transcripts in early pregnancy and at term.

OBJECTIVE: To demonstrate the expression of messenger RNA (mRNA) transcripts for placental leptin and leptin receptor in early gestation and at term, to quantitate transcriptional changes relative to stage of gestation, to localize transcripts within specific placental cell types, and to determine if transcripts also are expressed in cultured cells. METHODS: Expression of leptin and leptin receptor was assessed by reverse transcriptase-polymerase chain reaction, and leptin quantitated against a leptin mRNA competitor (MIMIC), in human placental villous tissue collected at term cesarean deliveries and earlier during gestation (7-14 weeks) upon elective terminations. In situ hybridization was used to identify cell types exhibiting transcripts for genes of interest. Additionally, tissue was dispersed enzymatically, cytotrophoblast cells progressed to syncytiotrophoblastic maturity in culture, and transcripts were assessed. RESULTS: Placental leptin and leptin receptor transcripts were identified in early (n = 6) and late (n = 5) gestation. Although no changes (P > .05) were apparent for receptor, leptin mRNA declined (P < .005) from (mean+/-standard error) 1.815+/-.491 attomoles/microg total RNA early in gestation to .013+/-.003 attomoles/microg total RNA at term. Leptin and leptin receptor transcripts were localized in trophoblast by in situ hybridization and were expressed in culture. CONCLUSION: Results suggest an ontogenetic decline in leptin mRNA with advancing gestation. Localization of leptin and leptin receptor transcripts in syncytiotrophoblasts, cells also responsible for the production of hormones vital to pregnancy maintenance, suggest a potential for autocrine or paracrine interactions within this tissue. Finally, transcript expression in cultured cells suggests the suitability of in vitro paradigms for future studies of leptin in pregnancy.

Adipose Tissue↗
PubMed2001

Quantitative RT-PCR luminometric hybridization assay with an RNA internal standard for cytokeratin-19 mRNA in peripheral blood of patients with breast cancer.

OBJECTIVES: To develop a highly sensitive quantitative RT-PCR hybridization assay for the determination of CK-19 mRNA in peripheral blood of patients with breast cancer. PATIENTS AND METHODS: Quantification of CK-19 mRNA was based on the coamplification of CK-19 mRNA with a recombinant CK-19 RNA internal standard (CK-19 RNA-IS) through RT-PCR. The biotinylated amplification products were immobilized on steptavidin coated wells, hybridized with digoxigenin labeled probes and determined through an antidigoxigenin antibody conjugated to alkaline phosphatase by luminometric detection. The developed luminometric hybridization assay was validated with samples containing total RNA of known amounts from CK-19 expressing cells (MCF-7) in the presence of 1 microg total RNA isolated from peripheral blood mononuclear cells (PBMC) of healthy controls and a constant amount of CK-19 RNA-IS. The method was applied for the quantitative determination of CK-19 mRNA in the peripheral blood of 26 healthy volunteers, 14 patients with stage IV breast cancer and 37 patients with stage I/II breast cancer before chemotherapy. RESULTS: Luminescence ratios for CK-19 mRNA and CK-19 RNA-IS were linearly related to the number of MCF-7 cells within the range of 1 to 2000 cells. The overall reproducibility of the assay (between-run) varied between 8.9% and 13.4%. The method can clearly detect CK-19 mRNA from 1 MCF-7 cell in the presence of 10(6) normal PBMC and is highly specific as none of the 26 healthy controls tested had detectable CK-19 mRNA levels, while 10 out of 14 (71.4%) and 9 out of 37 (24.3%) patients with stage IV and stage I/II breast cancer, respectively, were tested positive. CONCLUSION: The developed quantitative RT-PCR hybridization assay for CK-19 is reproducible, highly sensitive and specific, and can be used for a large-scale prospective evaluation of clinical samples.

Adolescent↗
PubMed1999

A patch coating method for preparing biocatalytic films of Escherichia coli.

A method has been developed for immobilizing viable but nongrowing Escherichia coli in highly uniform patches. The patches consist of a thin layer of bacteria in acrylate vinyl acetate covered with a thin layer of the same polymer devoid of bacteria and sealed by the edges. This method permits study of immobilized cell physiology in biocatalytic films by the assay methods used for suspended cells. Large numbers of patches of immobilized E. coli can be generated on metal or polyester sheets. Those described here are 12.7 mm in diameter; in them the cell layer is 30 microm thick and contains more than 5 x 10(8) viable cells. The method allows the cell-plus-polymer layer and the polymer sealant to be varied in thickness from 5 to 60 microm and from 7 to 80 microm, respectively. No leakage of cells was detected from 87% of the patches during 15 days of rehydration. Culturability of the immobilized cells, released by shaking the cells out of the porous polymer layer, was 80% of pre coating culturability. E. coli beta-galactosidase activity and measurements of total RNA and DNA from immobilized and suspended cells indicated that cells immobilized in the thin polymer layer have higher specific beta-galactosidase activity and a slower total RNA degradation rate than suspended cells over 15 days.

Bacterial Proteins↗
PubMed1987

Ribosome metabolism during the vitellogenic cycle of the mosquito, Aedes aegypti.

Ribosome accumulation and synthesis in the fat body of the mosquito Aedes aegypti increased by approx. 4-fold during 18 h after the blood meal, consistent with the pattern of total RNA accumulation during the synthetic phase of the vitellogenic cycle. By 24-30 h after the blood meal, the accumulated ribosomes began to be degraded, and the total RNA content in the fat body eventually returned to previtellogenic levels. A method has been developed for isolation of ribosomal subunits from fat body, and the ribosomal proteins have been shown to have properties similar to those from cultured Aedes albopictus cells by two-dimensional polyacrylamide gel electrophoresis. Proteins S1, S10, and A from the small ribosomal subunit are phosphorylated when fat body is incubated in Aedes saline in the presence of [32P]orthophosphate.

Adipose Tissue↗
PubMed2002

Expression and regulation by insulin of low-density lipoprotein receptor-related protein mRNA in human skeletal muscle.

Evidence suggests that increased hydrolysis and/or uptake of triglyceride-rich lipoprotein particles in skeletal muscle can be involved in insulin resistance. We determined the steady state mRNA levels of the low-density lipoprotein-related receptor (LRP) and lipoprotein lipase (LPL) in skeletal muscle of eight healthy lean control subjects, eight type 2 diabetic patients and eight nondiabetic obese individuals. The regulation by insulin of LRP and LPL mRNA expression was also investigated in biopsies taken before and at the end of a 3 h euglycemic hyperinsulinemic clamp (insulinemia of about 1 nM). LRP mRNA was expressed in human skeletal muscle (1.3+/-0.1 amol/microg total RNA in control subjects). Type 2 diabetic patients, but not nondiabetic obese subjects, were characterized by a reduced expression of LRP (0.8+/-0.2 and 1.3+/-0.3 amol/microg total RNA in diabetic and obese patients, respectively; P<0.05 in diabetic vs. control subjects). Insulin infusion decreased LRP mRNA levels in muscle of the control subjects but not in muscle of type 2 diabetic and nondiabetic obese patients. Similar results were found when investigating the regulation of the expression of LPL. Taken together, these results did not support the hypothesis that a higher capacity for clearance or hydrolysis of circulating triglycerides in skeletal muscle is present during obesity- or type 2 diabetes-associated insulin resistance.

Adult↗
PubMed1995

Accumulation of plasma cells in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits.

By screening a cDNA library constructed from aortic total RNA derived from Watanabe heritable hyperlipidemic (WHHL) rabbits by differential hybridization, we have obtained a cDNA encoding the kappa light chain of immunoglobulin. Northern blot analysis of total RNA prepared from aortas of WHHL and normal rabbits of various ages revealed that this light-chain mRNA accumulates gradually with age in aortas in WHHL rabbits. Northern blotting and in situ hybridization with an antisense oligonucleotide specific to rabbit immunoglobulin gamma heavy-chain mRNA also detected accumulation of this heavy-chain mRNA in advanced lesions of WHHL rabbit aortas. Moreover, immunohistochemical and electron microscopic analyses demonstrated the presence of plasma cells in the atherosclerotic lesions.

Amino Acid Sequence↗
PubMed2004

Rapid and specific detection of tdh, trh1, and trh2 mRNA of Vibrio parahaemolyticus by transcription-reverse transcription concerted reaction with an automated system.

Vibrio parahaemolyticus strains carrying the thermostable direct hemolysin (TDH) tdh gene, the TDH-related hemolysin (trh) gene, or both genes are considered virulent strains. We previously demonstrated that the transcription-reverse transcription concerted (TRC) method could be used to quantify the amount of mRNA transcribed from the tdh gene by using an automated detection system. In this study, we devised two TRC-based assays to quantify the mRNAs transcribed from the trh1 and trh2 genes, the two representative trh genes. The TRC-based detection assays for the tdh, trh1, and trh2 transcripts could specifically and quantitatively detect 10(3) to 10(7) copies of the corresponding calibrator RNAs. We examined by the three TRC assays the total RNA preparations extracted from 103 strains of Vibrio parahaemolyticus carrying the tdh, trh1, or trh2 gene in various combinations. The tdh, trh1, and trh2 mRNAs in the total RNA preparations were specifically quantified, and the time needed for detection ranged from 9 to 19 min, from 14 to 18 min, and from 9 to 12 min, respectively. The results showed that this automated TRC assays could detect the tdh, trh1, and trh2 mRNAs specifically, quantitatively, and rapidly. The relative levels of TDH determined by the immunological method and that of tdh mRNA determined by the TRC assays for most tdh-positive strains correlated. Interestingly, the levels of TDH produced from the strains carrying both tdh and trh genes were lower than those carrying only the tdh gene, whereas the levels of mRNA did not significantly differ between the two groups.

Automation↗
PubMed2006

A novel method to monitor the expression of microRNAs.

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18-25 nucleotides) with important roles in the regulation of gene expression. Although a large number of miRNAs have been identified in a variety of eukaryotic systems, the function of the vast majority of these molecules remains unknown. To study the functions of miRNAs, it is crucial to determine their spatial and temporal expression patterns. Although there are some existing methods that can analyze the expression of miRNAs, it is not an easy task for routine gene-expression studies. In this study, we have established a simple method to detect the expression of mature miRNAs. Total RNA was polyadenylated by poly(A) polymerase, and then cDNA was synthesized by a specific reverse transcriptase (RT) primer and reverse transcriptase using the poly(A)-tailed total RNA as templates. The expression of several mature miRNAs was assayed by this method. The expression profile of two miRNAs, determined by the polymerase chain reaction (PCR) assay, was identical to that determined by Northern blotting. All these data show that the poly(A)-tailed RT-PCR is a convenient method to detect the expression of miRNAs.

Blotting, Northern↗
PubMed1999

Characterization of early gene transcripts of molluscum contagiosum virus.

Molluscum contagiosum virus (MCV), a member of the family Poxviridae, replicates well in vivo but cannot be propagated in cell culture. The coding capacity of the MCV genome was previously determined by DNA nucleotide sequence analysis. The objective of the present study was to establish experimental systems for the identification and characterization of early MCV gene transcripts. MCV mRNA was obtained in three ways: (1) MCV early mRNA was synthesized in vitro using permeabilized virions, (2) MCV mRNA was extracted from MCV-infected skin tissue, and (3) MCV mRNA was extracted from MCV-infected human embryonic fibroblasts. RNA/DNA hybridization experiments showed significant early transcriptional activity in two parts of the MCV genome. Transcripts of 11 early MCV genes located in these parts of the genome, including two subunits of the MCV DNA-dependent RNA polymerase (mc077R and mc079R), the MCV poly(A)+ polymerase gene (mc076R), and the MCV MHC class I homolog (mc080R), were detected in reverse transcription-polymerase chain reaction experiments. Total RNA obtained from MCV-infected skin tissue was used to confirm these results. Three MCV early transcripts, mc002L, mc004.1L, and mc005L, produced distinct bands on rapid amplification of their 3' ends (3' RACE). The 5' mapping of transcription start sites of MCV open reading frames (ORFs) mc002L, mc004.1L, mc005L, and mc148R revealed that the MCV RNA polymerase transcription start sites are consistently located between 11 and 13 nucleotides downstream of the early MCV consensus promoter signal. When cDNA from both 5' and 3' mapping experiments was analyzed, MCV ORFs mc004. 1L and mc005L were found to be transcribed as a single bicistronic mRNA. The transcript from MCV ORF mc066L, encoding a glutathione peroxidase, was detected in in vitro synthesized MCV mRNA as well as in total RNA from MCV-infected human embryonic fibroblasts and MCV-infected skin. This indicates that despite the lack of an early MCV consensus promoter signal immediately proximal to the start codon, this particular gene is transcribed early during MCV infection.

Base Sequence↗
PubMed2002

Obtaining reliable information from minute amounts of RNA using cDNA microarrays.

BACKGROUND: High density cDNA microarray technology provides a powerful tool to survey the activity of thousands of genes in normal and diseased cells, which helps us both to understand the molecular basis of the disease and to identify potential targets for therapeutic intervention. The promise of this technology has been hampered by the large amount of biological material required for the experiments (more than 50 microg of total RNA per array). We have modified an amplification procedure that requires only 1 microg of total RNA. Analyses of the results showed that most genes that were detected as expressed or differentially expressed using the regular protocol were also detected using the amplification protocol. In addition, many genes that were undetected or weakly detected using the regular protocol were clearly detected using the amplification protocol. We have carried out a series of confirmation studies by northern blotting, western blotting, and immunohistochemistry assays. RESULTS: Our results showed that most of the new information revealed by the amplification protocol represents real gene activity in the cells. CONCLUSION: We have confirmed a powerful and consistent cDNA microarray procedure that can be used to study minute amounts of biological tissue.

Journal Article↗
PubMed1998

Cloning differentially regulated genes from chondrocytes using agarose gel differential display.

The technique of RNA differential display has been used extensively to clone differentially expressed genes from a wide variety of cells and tissues. Recently, a simplified method of cloning differential display products, separated on agarose gels, was described. Here we report an adaption of this method, using total RNA, to clone differentially expressed genes. The approach is simple and rapid, and requires only small quantities of total RNA. Utilising this approach, we have cloned three differentially regulated genes from chondrocytes stimulated to hypertrophy in vitro, and confirmed their pattern of expression by Northern blotting. These gene fragments were sequenced and found to correspond to known genes, although only one has previously been isolated from chondrocytes.

Animals↗
PubMed2005

[Effect of a novel splicing mutation (IVS2-2A-->C) of SEDL gene on RNA processing].

X-linked spondyloepiphyseal dysplasia tarda (SEDL) is a rare osteochondrodysplasia caused by the mutation of SEDL gene, which mainly involves vertebral bodies and hips. To explore the effect of the novel splicing mutation (IVS2 -2A-->C) of SEDL gene on mRNA processing in a large Chinese family with X-linked spondyloepiphyseal dysplasia tarda and to elucidate the molecular base of SEDL, total RNA was isolated from EDTA blood samples of patients and controls. RT-PCR was performed on total RNA. cDNA was analyzed by bi-directionally direct sequencing of PCR products and Polyacrylamide gel electrophoresis (PAGE). Sequencing analysis revealed that there were two kinds of cDNA in patients. One is that exon 2 directly spliced exon 4, that is, exon 3 absence from the mature mRNA; and the other is that exon 1 directly spliced exon 4, meaning both exon 2 and 3 being spliced out completely. Meanwhile one kind of cDNA that exon 1 directly spliced exon 3 was found in normal controls. By PAGE, RT-PCR amplified products, 679bp and 537bp, were detected in normal controls, while 567bp and 425bp fragments were found in affected individuals. Our data show that the mutation of the splice-acceptor site in intron 2 causes exon 3 entirely exclusion from the mature RNA transcripts in affected individuals. As the translation start site of the SEDL gene locates on exon 3, the splicing defect causes affected individuals failure to produce sedlin, which elucidates the causative role of SEDL gene in the pathogenesis of SEDL. The absence of exon 2 indicates that there is alternative splicing in SEDL gene. The alternative splicing was also found in normal controls, which demonstrated that the alternative splicing might not be related to the phenotype of SEDL. Because the alternative splicing of exon 2 occurred in the 5'UTR, it is not clear whether it affects the gene expression.

Base Sequence↗
PubMed1997

Two variants of quantitative reverse transcriptase PCR used to show differential expression of alpha-, beta- and gamma-fibrinogen genes in rat liver lobes.

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of alpha-, beta- and gamma-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (alpha-fibrinogen F = 14.64, P = 0.0003; beta-fibrinogen F = 3.74, P = 0.04; gamma-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

Animals↗