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At least 595 records · Page 33Linked to original sources
PubMed1979

Cell-free translation and thyroxine induction of carbamyl phosphate synthetase I messenger RNA in tadpole liver.

Total RNA of tadpole and frog (Rana catesbeiana) liver was isolated by either 7 or 8 M guanidine . HCl extraction and translated in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The identity of carbamyl phosphate synthetase I[carbamoyl-phosphate synthase (ammonia); ATP:carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5] synthesized in vitro with the purified enzyme was established as follows: (i) immunoprecipitation by a specific antibody; (i) comigration with purified carrier enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis; (iii) copurification with carrier enzyme by affinity chromatography on Cibacron Blue F3GA-coupled agarose; and (iv) formation of identical proteolytic cleavage products. Inclusion of protease inhibitors in the system resulted in no apparent change in the polypeptide molecular weight. These results indicate that carbamyl phosphate synthetase I is synthesized as a polypeptide that is indistinguishable from the mature enzyme by the analytical methods used and that it is not grossly modified during its transport into mitochondria. The level of translatable mRNA for carbamyl phosphate synthetase-I in tadpole liver was increased about 2-fold 1 day after thyroxine treatment and did not change significantly through 4 subsequent days of treatment. Thus the thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver is at least partly due to an increase of translatable mRNA for this enzyme.

Animals↗
PubMedDate not supplied

A method for isolation of RNA from Pneumocystis carinii.

Total RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and well behind the prokaryotic large rRNA species.

Animals↗
PubMed1989

Oestrogen receptor mRNA and a related RNA transcript in mouse ovaries.

Oestrogen receptor mRNA expression in mouse ovaries was analysed by Northern blotting of total RNA using 32P-labelled RNA probes complementary to different functional domains of the oestrogen receptor. The approximately 6.5 kb mouse oestrogen receptor mRNA transcript was present in immature and adult ovaries at extremely low abundance compared with uterus and oviduct. Using a probe complementary to the steroid-binding domain of the oestrogen receptor (probe EF), a novel RNA transcript of approximately 1.5 kb was also found in the ovaries but was absent from uterus and oviduct. The melting temperature of the hybrid produced by the approximately 1.5 kb transcript with probe EF was approximately 10 degrees C lower than that produced by authentic oestrogen receptor mRNA, which demonstrates incomplete sequence homology between the two transcripts and indicates that the approximately 1.5 kb RNA is not a truncated form of oestrogen receptor mRNA. Furthermore, the approximately 1.5 kb RNA lacks the DNA-binding domain found in the oestrogen receptor. The approximately 1.5 kb RNA, but not oestrogen receptor mRNA, was enriched in total RNA from isolated granulosa cells compared with residual ovarian tissue. The encoded product of this novel oestrogen receptor-related RNA could be a steroid-binding protein involved in oestrogen action in the ovaries.

Animals↗
PubMed1984

The synthesis of RNA and silk protein in Galleria mellonella silk glands.

The silk protein synthesis in silk glands of Galleria mellonella is preceded by the increase of total RNA content. The levels of the main RNA classes: 28S and 18S rRNA, tRNA as well as poly(A) + RNA change proportionally to the total RNA pool of glands. The fibroin-like silk protein of molecular weight of about 240 000 is characterized by the high content of four amino acids: glycine, alanine, serine and leucine, which account for more than 70% of amino acid residues. This fibroin-like protein is present in the posterior, middle and anterior parts of silk gland of the last instar larvae.

Animals↗
PubMed1992

Dietary selenium stabilizes glutathione peroxidase mRNA in rat liver.

The objective of these studies was to determine the step at which dietary selenium (Se) regulates the pre-translational expression of the gene for cytosolic Se-glutathione peroxidase (Se-GPX) in rat liver. Weanling male Sprague-Dawley rats were fed a Torula yeast-based Se-deficient diet, or the same diet supplemented with 0.5 mg/kg as Na2SeO3, for at least 40 d. Livers were excised and divided into three portions for isolation of nuclei, for purification of total RNA and for assay of Se-GPX activity. Nuclei were used in run-on transcription assays and for isolation of total nuclear RNA. Nuclear RNA and total liver RNA were probed with segments from a rat Se-GPX cDNA in Northern blot and slot blot assays. Despite marked differences in Se-GPX activity, there were no significant differences between dietary groups in the transcription rates of the Se-GPX gene. Species hybridizing to Se-GPX were not detected in nuclear RNA. However, steady state levels of Se-GPX mRNA were markedly reduced by Se deficiency. These results suggest that dietary Se exerts its effect on pretranslational Se-GPX gene expression at the level of cytosolic mRNA stabilization.

Animals↗
PubMed1979

Transcription of viral genes in chromatin from adenovirus 2 transformed cells by exogenous eukaryotic RNA polymerases.

The transcription of chromatin from adenovrius 2 transformed rat cells by murine plasmacytoma RNA polymerases I, II and III has been studied. Both the total RNA synthesis and transcription of the integrated adenovirus 2 genes by RNA polymerase II represent de novo DNA transcription as assessed by their sensitivity to actinomycin D. It is shown that each RNA polymerase class has characteristic ionic strength activation profiles and metal ion requirements. RNA polymerase II transcribes the integrated adenovirus 2 genes in chromatin at a frequency 25- to 50-fold higher than their sequences are represented in the genome. In contrast, no detectable viral RNA is synthesized when deproteinized DNA is transcribed. In the presence of Mn2+, all three RNA polymerases (I, II and III) transcribe the integrated viral genes at approximately the same relative frequencey. However, the Mg2+ as divalent cation, the proportion of the total RNA which represents viral gene transcripts is increased 3- to 4-fold with RNA polymerase II, while it remains unchanged for RNA polymerases I or III.

Adenoviridae↗
PubMed2004

Flotillin-1 in the substantia nigra of the Parkinson brain and a predominant localization in catecholaminergic nerves in the rat brain.

The substantia nigra cells of a normal and Parkinson's disease human brain were obtained by the micropunch procedure and total RNA was isolated. Differential display RT-PCR of the total RNA revealed differentially expressed cDNAs that were identified by sequencing. This resulted in the identification of a panel of known and unknown differentially expressed genes. Complex I (NADH ubiquinone oxidoreductase) and Complex IV (cytochrome oxidase) whose expressions are decreased in Parkinson's disease were reduced in the Parkinson brain. Of the various differentially expressed genes, flotillin-1, also known as reggie-2, was of great interest to us. It is a relatively new protein which is an integral membrane component of lipid rafts and has been implicated in signal transduction pathway events. In situ hybridization histochemical studies with human and rat brain sections revealed the presence of this mRNA in discrete neuronal (and possibly glial) cells of the substantia nigra, locus coeruleus, cortex, hippocampus, hypothalamus, thalamus, motor nuclei, nucleus basalis, raphe nucleus, and other brain regions. Immunohistochemical studies revealed that flotillin-1 is not present in all the regions where the message was found. In the rat brain, the most prominent observation was the revelation of all catecholamine cells (dopamine, norepinephrine, epinephrine) by the flotillin-1 antibody (1:100 dilution). At a more concentrated dilution (1:10) other neuronal cells (e.g., cortex, thalamus, hindbrain) were observed. At both dilutions dense dopaminergic fibers were observed in the rat caudate-putamen, nigrostriatal tract, and substantia nigra. It is significant that there is an increased gene expression of flotillin-1 in the Parkinson substantia nigra/ventral tegmental area. The role of flotillin in these cells is unclear although it is interesting that the reggie-2/flotillin-1 gene was upregulated during retinal axon regeneration in the goldfish visual pathway (Schulte et al., Development 124:577-87, 1997) which suggests that flotillin-1/reggie-2 might play a role in axonal growth from the remaining substantia nigra cells of the Parkinson brain.

Aged↗
PubMed2003

A methodological approach to investigate steady state fucoxanthin chlorophyll a/c binding protein mRNA levels in Wadden Sea sediments.

A method was established to investigate the steady state levels of mRNAs from genes encoding fucoxanthin chlorophyll a/c binding proteins (Fcp) of diatoms in situ. During the study, which was performed with Wadden Sea sediments from the German North Sea shore near Dangast, oxygenic photosynthesis was carried out mainly by pennate diatoms. Field samples were taken after tidal exposure from dawn up to late afternoon at 2-hourly intervals, and frozen in liquid nitrogen. In the laboratory, total RNA was isolated by isopycnic ultracentrifugation in caesium chloride gradients. Yields of approximately 10-300 micro g RNA per gram wet sediment were obtained. Defined amounts of total RNA were blotted onto nylon membranes and hybridised with probes against the fcp2 and 18S rDNA genes of Cyclotella cryptica. To estimate the steady state amount of fcp mRNAs, fcp signal intensities were normalized to the signal intensities obtained from hybridisation to an 18S rDNA gene probe. In the two time-course studies performed to demonstrate the applicability of the method, the steady state levels of fcp mRNA increased up to 12-fold with the onset of light, reaching a maximum 6-8 h after sunrise before they decreased again. Possible reasons for this time-course are discussed.

Animals↗
PubMed2003

Regulation of inhibin alpha- and beta(A)-subunit messenger ribonucleic acid levels by gonadotropins and IGF-I in cultured chicken granulosa cells.

A quantitative competitive reverse transcription polymerase chain reaction assay (QC RT-PCR) for quantifying the absolute levels of the expression of inhibin alpha- and beta(A)-subunits in chicken granulosa cells showed that these subunits are expressed in different amounts depending on follicular maturation. The present study determined the regulation of the expression of these subunits. The individual effect of different doses of IGF-I, LH or FSH (1-100 ng/ml) or the combination of IGF-I with either LH or FSH at different concentrations, on the expression of inhibin alpha- and beta(A)-subunit was determined on cultured granulosa cells of F(1) and the combined F(4)+F(5) follicle. Cells were cultured for 48 h in 6-well plates with or without added hormones. Culture medium was discarded, cells were washed and total RNA was extracted from the cells. Five hundred nanograms of total RNA was reverse transcribed using specific primers and coamplified with an internal standard, as described previously, to determine expression level in the cells. IGF-I, LH, and FSH enhanced the inhibin alpha-subunit mRNA levels in a dose dependent manner in both F(1) and the combined F(4)+F(5) whereas inhibin beta(A)-subunit was not affected. The effects of FSH, LH were more expressed in F(1) follicles compared to F(4)+F(5) on the alpha-subunit. The addition of IGF-I and either LH or FSH during the culture period significantly increased the stimulatory effects of both LH and FSH on the expression of inhibin alpha-subunit in F(1) follicles but had no significant effect on the inhibin beta(A)-subunit. The results suggest that the changing expression levels of inhibin alpha-subunit during follicular development are the result of the regulatory effect of the interaction between IGF-I and the gonadotropins and that the regulation of this subunit may be the main factor for the regulation of the protein inhibin levels. Other factors may be also implicated in the changing expression levels of the beta(A)-subunit.

Animals↗
PubMed1994

Effect of retinoic acid on mucin gene expression in rat airways in vitro.

Ultrastructural examination of rat tracheal explants at various times of culture in a serum-free and hormone-supplemented medium containing retinoic acid showed that the cytological characteristics of the epithelium were well preserved for at least 192 h. Hybridization analyses for mucin core protein mRNA in the explants were performed with a 30-base oligonucleotide probe, the design of which was based on the tandem repeat sequence of the rat intestine mucin core protein. The probe reacted with total RNA prepared from trachea, intestine and colon, but not with total RNA obtained from liver or alveolar region of the lung. Type-I keratin expression was observed in the explant grown at different periods of time in a medium with and without retinoic acid. The hybridization probe gave a prominent reaction with RNA preparations obtained from tracheal explants incubated for as long as 192 h in a medium containing retinoic acid. In the absence of retinoic acid, however, the mucin message was evident at the 24 h time point but thereafter decreased to barely detectable levels. When retinoic acid was added at 96 h to the latter cultures, the mucin mRNA was prominent again after additional incubation for 24 and 48 h. Northern-blot analyses of tracheal RNA showed a diffuse band at approx. 7.5 kb. Addition of a variety of chemical and pharmacological agents to explants cultured in the presence of retinoic acid had no dramatic induction or inhibitory effects on the mucin mRNA. Only the steroid prednisolone had a reproducible inhibitory effect.

Animals↗
PubMed1999

Human dermal microvascular endothelial cells express inducible nitric oxide synthase in vitro.

Stimulation of inducible nitric oxide synthase with subsequent release of nitric oxide in large amounts may play a critical part either in host defense reactions or in the pathophysiology of the inflammatory response syndrome leading to septic shock. The aim of the present study was to investigate whether human dermal microvascular endothelial cells exhibit the typical characteristics of an inducible nitric oxide synthase expressing cell. A strong effect on inducible nitric oxide synthase gene expression could be detected when the cells were coincubated with the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha with inducible nitric oxide synthase cDNA concentrations averaging 11.7 +/- 0.6 amol per microg total RNA at 24 h, and 25.0 +/- 1.4 amol per microg total RNA at 48 h, respectively. Intracellular staining with an antibody recognizing inducible nitric oxide synthase protein and subsequent analysis by flow cytometry revealed a 4-fold increase of inducible nitric oxide synthase protein in human dermal microvascular endothelial cells treated with interferon-gamma/tumor necrosis factor-alpha. This was accompanied by a significant elevation in nitrite/nitrate concentrations in the cell-free culture supernatants. Our results indicate that human dermal microvascular endothelial cells are provided with an inducible nitric oxide synthase system and can be regarded as an appropriate cell model for investigating inducible nitric oxide synthase gene expression and nitric oxide properties in microvascular endothelial cells.

Cells, Cultured↗
PubMed2006

Molecular biology of ovarian aromatase in sex reversal: complementary DNA and 5'-flanking region isolation and differential expression of ovarian aromatase in the gilthead seabream (Sparus aurata).

To elucidate the involvement of aromatase in sex reversal, the gilthead seabream ovarian P450 aromatase (cyp19a1a) cDNA and its 5'-flanking region were isolated and characterized. Northern blot analysis revealed that only one cyp19a1a transcript (2.0 kb) is expressed in the ovary. Four cAMP-responsive elements were identified at the 5'-flanking region of seabream cyp19a1a indicating a high potential to respond to gonadotropin signaling. Studying the seasonal profile, two expression peaks of cyp19a1a transcripts in the ovarian tissues were found in July (about 15000 copies/ng total RNA) for ambisexual fish and in December (about 12000 copies/ng total RNA) for spawning females. Starting from September, transcript levels of cyp19a1a in the ovarian portions of the male-developing gonads gradually decreased. Furthermore, the ovarian portions of the female gonads expressed cyp19a1a at a significantly higher level than the ovarian portions of the male gonads after November. Taken together with levels of plasma estradiol in reversing females being significantly higher than those in developing males, the above results reinforce the importance of cyp19a1a in sex reversal. In vitro exposure of ovarian fragments to gonadotropins (hCG) at 1, 10, and 100 IU/ml significantly (P < 0.05) upregulated cyp19a1a expression. Additionally, expression of cyp19a1a displayed a stronger and significant correlation with the transcript expression of ovarian Lh receptor rather than Fsh receptor during the ambisexual stage. Our results indicate that the differential expression of cyp19a1a gene is associated with sex reversal and that gonadotropin signals (particularly Lh) may serve as major players in regulating the expression of cyp19a1a during the process of sex reversal.

5' Flanking Region↗
PubMed1997

Increased bax and interleukin-1beta-converting enzyme messenger ribonucleic acid levels coincide with apoptosis in the bovine corpus luteum during structural regression.

Recent reports have demonstrated that prostaglandin F2alpha-induced generation of reactive oxygen species or their intermediates inhibits progesterone synthesis and may also serve as a trigger for apoptosis in the corpus luteum (CL). BCL-2, an inhibitor of apoptosis in a wide variety of cell types, has been reported to prevent oxidative stress-induced cell death. Thus, the present studies were conducted to determine whether levels of mRNA encoding BCL-2 and related members of this gene family (BAX and BCL-Xshort, which induce apoptosis; BCL-Xlong, a BCL-2 homologue that prevents apoptosis) differed in functional (Day 21 of pregnancy) versus regressed (Day 21 of the estrous cycle) CL in the bovine ovary. Levels of mRNAs encoding p53, a transcriptional regulator of the bcl-2 and bax genes, and interleukin-1beta-converting enzyme (ICE), a protein recently implicated in the induction of apoptosis whose expression may be enhanced by oxidative stress, were also assessed. Partial cDNA clones encoding bovine bax, bcl-x, p53, and Ice were isolated using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with total RNA prepared from functional or regressed CL. A bovine bcl-2 cDNA could not be isolated from luteal tissue RNA despite the use of several primer pairs for amplification. Total RNA was then extracted from functional or regressed CL and analyzed by Northern blot analysis. The occurrence of apoptosis in regressed CL, as evidenced by the presence of internucleosomal DNA cleavage, was associated with a significant increase in both bax and Ice mRNA levels as compared with levels of bax and Ice expression in functional CL (p < 0.05, n = 3). There were no significant differences in bcl-x or p53 mRNA levels in functional versus regressed CL. Analysis of bcl-x mRNA by RT-PCR revealed that the long form was the primary, if not only, mRNA expressed in functional and regressed bovine luteal tissue. On the basis of data that increased expression of bax is associated with, and may be required for, apoptosis in ovarian granulosa cells and germ cells, we propose that BAX may play a similar role in apoptosis induction during luteal regression. Moreover, the increased Ice mRNA levels in regressed CL provides the first evidence that the ICE family of death proteases may be involved in luteolysis.

Animals↗
PubMed1995

Expression studies on four members of the pMGA multigene family in Mycoplasma gallisepticum S6.

A large family of related genes known as pMGA exists in the avian pathogen Mycoplasma gallisepticum but only a single member of this family was previously found to be expressed in one strain of this bacterium. In this work two unrelated strains of M. gallisepticum were also shown by amino-terminal sequencing to express a unique pMGA polypeptide in both cases. To investigate pMGA gene selection in M. gallisepticum, mRNA expression was analysed in M. gallisepticum strain 56 using reverse transcription-PCR (RT-PCR) and Northern blot techniques with probes for several members of the pMGA multigene family. It was shown that the pMGA message is 2.2 kb in size and is monocistronic. RT-PCR detected four different pMGA mRNA molecules but their relative yields were significantly affected by magnesium concentration. By quantitative Northern analysis, the relative abundances of the four pMGA mRNAs in M. gallisepticum S6 total RNA was determined: the pMGA1.1 mRNA predominated [1.88 ng (micrograms total RNA)-1] but at least three other pMGA genes were found to be transcribed but at much lower levels (20 to 40-fold lower). The pMGA1.1 mRNA is expressed at a level five times higher than the tuf gene, known to be one of the most abundantly expressed proteins in the prokaryotic cell. The start point of transcription for pMGA1.1 was determined and probable promoter assigned. From these data it appears likely that transcriptional control plays a major role in the selection of pMGA gene expression in the M. gallisepticum cell.

Amino Acid Sequence↗
PubMed1990

Developmental profile of striatal preprotachykinin gene expression.

The normal developmental profile of preprotachykinin (PPT) gene expression was determined in rat striatum from embryonic day 20 to adulthood (greater than 45 days of age). At embryonic day 20, the amount of striatal PPT mRNA was approximately 8% of adult levels, and this specific mRNA approached adult levels by postnatal days 12-15. The absolute amount of PPT mRNA, determined by comparison with PPT mRNA synthesized in vitro, ranged from 0.2 pg/micrograms of total RNA at embryonic day 22 to 5-6 pg/micrograms of total RNA in adult striata. In addition, the proportion of the various PPT mRNAs derived from the same primary transcript by alternate splicing was determined in the developing animal. At those ages at which PPT mRNA levels were significantly less than those in the adult, there was a slight (10%) but statistically significant increase in the relative amount of gamma-PPT mRNA with respect to the amount of beta-PPT mRNA. Because these mRNA species encode different combinations of tachykinin peptides, these data suggest that the proportion of substance P versus various neurokinin A-related peptides may be developmentally regulated.

Aging↗
PubMed2001

PKCalpha and PKCgamma overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line.

PURPOSE: The overexpression of PKCalpha or PKCgamma for extended periods of time causes the formation of lentoid bodies in the N/N 1003A rabbit lens epithelial cell line. To determine how differentiated the lentoid bodies are, we have looked for alphaA-, alphaB-, beta-, and gamma-crystallin levels in lentoid bodies after 4 and 8 weeks of lentoid body development. METHODS: Cells overexpressing PKCalpha or PKCgamma were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid bodies were fixed and stained with PKCalpha or PKCgamma antibodies along with either alphaA-, alphaB-, beta-, or gamma-crystallin antisera and viewed under a confocal microscope. Lentoid bodies were harvested in lysis buffer and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was probed with either alphaA-, alphaB-, beta-, or gamma-crystallin antibodies for 12 h. Total RNA from lentoid bodies was isolated and 5 microg of total RNA was transcribed to first-strand cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis. RESULTS: alphaB-crystallin was present in normal N/N 1003A cells and the lentoid bodies formed from PKCalpha and PKCgamma overexpression. alphaA-crystallin was only detectable in lentoid bodies after PKCalpha or PKCgamma overexpression. RT-PCR was able to detect beta-crystallin expression while the Western blot analysis and immunocytostaining detected small amounts of beta-crystallin protein. No gamma-crystallin expression was noted in these lentoid bodies. CONCLUSIONS: Overexpression of PKCalpha or PKCgamma in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not only alphaB-crystallin but alphaA- and beta-crystallin. These results suggest a role for PKCs in lens epithelial cell differentiation to a fiber cell.

Animals↗
PubMed1992

Regulated expression of syndecan in vascular smooth muscle cells and cloning of rat syndecan core protein cDNA.

cDNA encoding the core protein of rat syndecan was cloned from a neonatal rat aortic cDNA library by polymerase chain reaction amplification. Expression of syndecan mRNA in rat aortic vascular smooth muscle (VSM) cells was demonstrated by reverse transcriptase-linked polymerase chain reaction amplification of syndecan sequences using total RNA from rat aortic VSM cells as templates. Polyclonal antibodies against rat syndecan core protein were produced by immunizing rabbits with a recombinant fusion protein containing a fragment of the extracellular domain. The anti-syndecan antibodies immunoprecipitated a large 35SO4-labeled molecule synthesized by cultured rat aortic VSM cells. The immunoprecipitated molecule was identified as a hybrid proteoglycan, based on results of alkaline, nitrous acid, and chondroitinase ABC digestions. On immunoblots the antibodies recognized a proteoglycan of greater than 200 kDa, with a core protein size after deglycosylation of approximately 50 kDa. The anti-syndecan antibodies stained cultured rat aortic VSM cells as well as tissue sections of neonatal and adult rat aortas in the medial, smooth muscle layer. On Northern blots of RNA isolated from cultured VSM cells, a syndecan cDNA probe hybridized to a major RNA species of 2.6 kilobases. Quantitative Northern blot analysis of total RNA isolated from VSM cells harvested at different cell densities revealed a decrease in syndecan mRNA levels with increased cell density. These results demonstrate the regulated synthesis of syndecan by rat VSM cells.

Amino Acid Sequence↗
PubMed1995

Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals.

The major bcr-abl fusion gene is presently seen as the hallmark of chronic myeloid leukemia (CML) and presumably as the cause of its development. Accordingly, long-term disappearance of bcr-abl after intensive therapy is considered to be a probable cure of CML. The nested reverse transcriptase-polymerase chain reaction (RT-PCR) provides a powerful tool for minimal residual CML detection. The RT-PCR was optimized by (1) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells, (2) using a specific abl primer in this reverse reaction, and (3) reamplifying 10% of the RT-PCR product in nested amplification. This optimized RT-PCR permitted us to detect up to 1 copy of RNA bcr-abl synthesised in vitro, mixed with yeast RNA in an equivalent quantity to 10(8) white blood cells (WBCs). Using this highly sensitive RT-PCR during the follow-up of CML patients, a signal was unexpectedly found in healthy controls. Therefore, a systematic study of the possible expression of bcr-abl RNA in the WBCs of healthy adults and children and in umbilical cord blood was undertaken. It showed the presence of bcr-abl transcript in the blood of 22 of 73 healthy adults and in the blood of 1 of 22 children but not in 22 samples of umbilical cord blood.

Adolescent↗