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C-erbB-2-oncogene expression in breast carcinoma: analysis by S1 nuclease protection assay and immunohistochemistry in relation to clinical parameters.

The c-erbB-2 mRNA was detected by the S1 nuclease protection assay and Northern blotting in breast cancer tissues. In contrast to the Northern blot analysis which has been used in all recent publications concerning c-erbB-2 expression on the level of RNA, the S1-nuclease protection assay has distinct advantages with respect to sensitivity, reproducibility, and handling of radioactive probes. We compared the expression of c-erbB-2 in 120 breast carcinomas which were operated in the years 1989-1990 on the level of the mRNA (S1 nuclease protection assay) and the protein (immunohistochemistry), respectively. In general, results obtained with both methods were in good agreement. Only minor differences in classification were observed with 18 samples, all of them belonging to either the moderate or the weak c-erbB-2 expression phenotype. In addition, the level of c-erbB-2-protein was investigated by immunohistochemistry in 271 breast carcinomas which were operated in the years 1984-1987. Comparison of the level of c-erbB-2 expression with the patient history indicates that patients whose tumors had already metastasized to the axillary nodes showed a reduced overall and disease-free survival in the group with pronounced expression of the c-erbB-2 protein. Thus the strong expression of c-erbB-2 oncogene in primary breast cancers appears to be an additional and particular prognostic factor in lymph node positive patients.

Blotting, Northern↗

Use of a multiple S1 nuclease protection assay to monitor changes in RNA levels for type 1 phosphatase and several proto-oncogenes in response to insulin.

Changes in insulin-regulated gene expression occur in a time- and tissue-dependent fashion. To monitor these changes we have adapted the S1 nuclease protection assay to allow simultaneous estimation of multiple RNA species in a single sample by using synthetic oligonucleotides of various lengths as probes for specific RNA species, which can then be resolved by electrophoresis. The multiple S1 nuclease protection assay was used to assess the influence of insulin on the RNA concentrations of 12 different genes in human skeletal muscle. Estimates obtained by this assay were comparable with those obtained by Northern analysis. RNA levels for proto-oncogene c-src displayed a transient 4-fold increase, whereas RNA levels for type 1 protein phosphatase were suppressed by 50% during the same time period. RNAs corresponding to known insulin-responsive genes such as c-fos, c-myc, c-Ha-ras, and c-src displayed rapid and transient 2-4-fold increases between 30 and 60 min as detected by either Northern analysis or the multiple S1 nuclease protection assay. In addition, RNA levels for the insulin receptor, Glut-4, Glut-3, and c-jun were apparently unaffected by exposure of the cells to insulin.

Antisense Elements (Genetics)↗

Solution hybridization-nuclease protection assays for sensitive detection of differentially spliced substance P- and neurokinin A-encoding messenger ribonucleic acids.

In this chapter we discussed methods that can be used for the sensitive detection and quantitation of differentially or alternatively spliced mRNAs as well as mRNAs of low abundance. Although mechanisms responsible for splicing (and differential splicing in particular) have not been fully determined, many RNAs derived from a variety of genes have been observed to undergo the process. The impact of splicing with regard to the expanded potential of gene expression emphasizes the usefulness of the solution hybridization-nuclease digestion technique described here, compared to Northern blot analysis. The use of radiolabeled cRNA(s) provides for an assay of both high specificity and high sensitivity. While end-labeled cDNA probes can be used, they do not have the sensitivity inherent in the assay performed with uniformly radiolabeled cRNAs. If multiple mRNAs are derived from a single gene as a result of differential or alternative precursor RNA splicing, however, the results with a cRNA probe may initially appear to be quite complicated, and end-labeled cDNAs may yield more easily interpretable results. Nonetheless, both types of probes are useful in the context of gene expression analysis, and it is clear that for routine purposes of quantitation cRNA probes in solution hybridization-nuclease protection assays are clearly more desirable than RNA blot analyses due to their truly quantitative nature as well as ease of assay.

Autoradiography↗

Expression of GABA(A) receptor isoform genes in the cerebral cortex of cirrhotic and alcoholic cases assessed by S1 nuclease protection assays.

Pathogenic processes underlying the localized reduction in neuronal number in cerebral cortex in human alcoholics have been reported to be associated with selective variations in the parameters of GABA(A) receptor site binding. Since the properties of the receptor complex depend on its isoform composition, we studied how the expression of GABA(A) receptor subunit isoform genes varied with alcoholism. Cerebral cortex tissue was obtained at autopsy from chronic human alcoholics (average ethanol intake > 80 g/day for most of their adult lives; n = 17) and matched controls (< 20 g/day ethanol; n = 15). Eight of the alcoholics and five of the controls had pathologically confirmed cirrhosis of the liver. Expression of alpha1, alpha2, alpha3, alpha5, beta1, beta3, and gamma2 GABA(A) mRNA was assessed by S1 nuclease protection assays. After phosphorimager quantitation and normalization to GAPDH mRNA and 18S rRNA, none of the mRNA species showed significantly different expression in uncomplicated alcoholics. Analysis of differences in the patterns of expression of the various subunits showed the alpha1 signal was strongest in combined cirrhotic motor cortex while the alpha3 and beta3 values were greatest in combined cirrhotic frontal cortex. It appears that only major differences in mRNA expression may be detected by this technique in human brain.

Adult↗

The quantitative analysis of multiple mRNA species using oligonucleotide probes in an S1 nuclease protection assay.

The quantitative measurement of steady-state mRNA levels is fundamental to the analysis of gene expression. A variety of techniques are widely used to achieve this including Northern blotting, RNase protection, and S1 nuclease protection. We describe here in detail a relatively recent extension of the S1 nuclease protection technique (1) in which radiolabeled oligonucleotides are used as probes in a solution hybridization assay (2). The principle advantage of this technique is that it allows, in a single RNA sample, the simultaneous measurement of the relative levels of at least six mRNA species, including that of a control mRNA. Further, a large number of RNA samples can be analyzed at one time.

Nucleic Acid Hybridization↗

Fibroblast growth factor (FGF)-2 sense and antisense mRNA and FGF receptor type 1 mRNA are present in the embryonic and adult rat nervous system: specific detection by nuclease protection assay.

In the present study, we attempted to clarify the controversial question whether basic fibroblast growth factor (bFGF, FGF-2) mRNA is present or absent in the embryonic central nervous system (CNS). For this purpose we analyzed the expression of the FGF-2 mRNA in the embryonic and adult forebrain, brainstem, and spinal cord using the highly specific ribonuclease protection assay. Using this method we were able to detect FGF-2 mRNA in the rat CNS of embryonic day (E) 16 and 17, however, at lower levels compared to adult FGF-2 mRNA levels. In addition, we show that a FGF-2 antisense transcript is expressed in embryonic CNS tissue. Furthermore, using this method, we demonstrate FGF receptor 1 mRNA in the rat embryonic and adult CNS. The presence of FGF-2 and FGF receptor 1 suggests a physiological role for this growth factor during the development of the embryonic CNS.

Aging↗

Regional distribution and developmental expression of epidermal growth factor and transforming growth factor-alpha mRNA in mouse brain by a quantitative nuclease protection assay.

A solution-hybridization ribonuclease-protection assay was used to identify epidermal growth factor (EGF) mRNA in mouse brain and to compare the regional and developmental levels of EGF gene expression in the CNS with those of its structural homolog, transforming growth factor-alpha (TGF-alpha). Adult brain regions examined included brainstem, cerebellum, cerebral cortex, hippocampus, basal hypothalamus, olfactory bulb, olfactory tubercle, striatum, and thalamus. While both EGF and TGF-alpha mRNAs were detected in all regions, TGF-alpha mRNA levels were 15-170 times higher, ranging from 0.39 (cerebellum and cerebral cortex) to 2.93 (striatum) pg TGF-alpha mRNA/micrograms total cytoplasmic RNA. In contrast, EGF mRNA levels ranged from 11 to 36 fg EGF mRNA/micrograms, with the highest regional concentrations observed in olfactory bulb, basal hypothalamus, and cerebellum. In our comparison between sexes, no significant male-female differences in EGF or TGF-alpha mRNA levels were observed for any region of adult brain. However, in the pituitary gland, consisting of both endocrine and neural elements, EGF and TGF-alpha mRNA levels were significantly higher in males (234 and 215 fg/micrograms, respectively) than in females (172 and 118 fg/micrograms, respectively). An examination of growth factor gene expression in the developing CNS revealed EGF and TGF-alpha mRNAs detectable as early as embryonic day 14 (earliest time point studied). While gene expression for both peptides continued into the postnatal period, EGF and TGF-alpha mRNA levels were nearly equal to adult concentrations by postnatal day 10. Taken together, our findings provide evidence for the synthesis of EGF in brain and suggest a role for both EGF and TGF-alpha in the development and support of the mammalian CNS.

Aging↗

Expression of a gene for mouse eucaryotic elongation factor Tu during murine erythroleukemic cell differentiation.

The eucaryotic elongation factor Tu (eEF-Tu) is a single polypeptide with an approximate Mr of 53,000. During protein synthesis eEF-Tu promotes the binding of aminoacyl-tRNA to the ribosome. To study the expression of the gene(s) for this factor, a genomic clone was isolated that contains a mouse eEF-Tu gene. We screened a phage genomic library with a synthetic oligonucleotide probe complementary to a region of the Saccharomyces cerevisiae and Artemia sp. eEF-Tu genes which codes for an area that is highly conserved between both yeast and Artemia sp. eEF-Tu. From approximately 75,000 phage plaques we obtained five isolates with apparently identical inserts. All five clones contained a 3.8-kilobase EcoRI fragment that hybridized to additional oligonucleotide probes corresponding to different conserved regions of eEF-Tu. We sequenced the 5' end of one genomic clone and determined the length of the cloned fragment that was protected by eEF-Tu mRNA in S1 nuclease protection assays. A quantitative S1 nuclease protection assay was used to compare the relative steady-state levels of eEF-Tu mRNA in total mRNA in total RNA isolated from hexamethylene-bisacetamide-induced murine erythroleukemia cells. The results show a dramatic reduction in the steady-state level of eEF-Tu mRNA as differentiation proceeds. A similar reduction in transcription of eEF-Tu mRNA was observed in isolated nuclei. Finally, we examined the in vivo synthesis of eEF-Tu during differentiation and found that it declined in a manner parallel to the decline in the steady-state level of eEF-Tu mRNA. In addition, we have isolated and sequenced a cDNA clone for mouse eEF-Tu. The derived amino acid sequence is compared with sequences from other eucaryotes.

Animals↗

Insulin regulation of multiple ribonucleic acid species in human skeletal muscle in insulin-sensitive and insulin-resistant subjects.

In vivo short term (2 h) insulin-regulated gene expression was examined in skeletal muscle of persons with differing insulin sensitivities. Nine genes were analyzed by a S1 nuclease protection assay with multiple probes (multiple S1 nuclease protection assay) to allow the simultaneous examination of RNA abundances from the multiple genes. In insulin-sensitive individuals, 5 of these 9 genes were insulin responsive. RNA from the proto-oncogenes c-Ha-ras, c-myc, and c-src transiently increased 2- to 4-fold within 30 min of insulin infusion. In addition, the RNA abundance of myf-5, a muscle specific differentiation factor, increased 3-fold with a time course similar to that of c-Ha-ras, c-myc, and c-src. In contrast, type 1 protein phosphatase alpha (PPP1A) RNA levels decreased by 50% within 30 min. In insulin-resistant individuals, the RNA levels of c-Ha-ras and myf-5 did not increase, whereas c-src RNA did increase within 30 min of insulin infusion. RNA encoding c-myc transiently increased in both groups; however, this response was lower in insulin-resistant individuals than in insulin-sensitive individuals in a pattern similar to c-Ha-ras and myf-5. PPP1A RNA levels slightly increased in insulin-resistant individuals. In both insulin-sensitive and insulin-resistant persons, RNA quantities of GLUT4, c-jun, c-fos, and the insulin receptor did not change over the period of insulin infusion. However, overall RNA levels of the insulin receptor and c-jun were lower in insulin-resistant individuals.

Adolescent↗

Brain-specific polyA- transcripts are detected in polyA+ RNA: do complex polyA- brain RNAs really exist?

Transcripts encoded by 2 different rat genomic clones, rg13 and rg100, appear to be typical brain-specific polyA- RNAs, as defined by previous criteria (rare, polysomal, and postnatally expressed from single-copy genes). However, we have found by using a sensitive nuclease protection assay that low levels of these transcripts (10% and 3%, respectively) are detected in polyA+ RNA. To determine if rg transcripts that fractionate as polyA- could have resulted from nicking of polyA+ RNA, we assessed the integrity of 2 known polyA+ RNAs, those of tyrosine hydroxylase, a 2-kilobase (kb) mRNA, and sodium channel, a 9.5-kb RNA. Using RNA prepared by several different procedures, including LiCl-urea and guanidine thiocyanate followed by CsCl centrifugation, the shorter message fractionated as polyA+ after 2 cycles over oligodeoxythymidine (oligo-dT) cellulose, whereas the majority of the longer sodium channel RNA fractionated as polyA, as assayed by nuclease protection using probes from the 5' end of the 2 genes. However, on Northern blots, the same RNA preparations showed an intact 9.5-kb sodium channel band only in polyA+ RNA, suggesting that the polyA- RNAs were randomly cleaved, resulting in a smear of sizes that could not be detected as a discrete band. These data imply that long messages may be nicked during standard isolation procedures and that this would not be detected by Northern blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

Improved quantification with validation of multiple mRNA species by polymerase chain reaction: application to human myocardial creatine kinase M and B.

OBJECTIVES: Quantitation by polymerase chain reaction (PCR) has not been validated by an independent assay and is not easily applicable to multiple samples. The aim of this study was therefore to attempt to quantify human heart creatine kinase M (CKM) and B (CKB) mRNA simultaneously and to validate the method with a quantitative S1 nuclease protection assay. METHODS: Conditions were optimised to achieve high efficiency of reverse transcription and PCR amplification of a short target sequence using total human heart RNA and an in vitro transcribed RNA standard as a substrate. A nested radiolabelled primer was used for specific detection and quantification of the amplified DNA sequence. RESULTS: The amplification efficiency for CKM and CKB mRNA were 0.98 (SD 0.07) and 1.11 (0.04) respectively. CKM and CKB mRNA levels were determined in 42 samples from 24 human hearts and found to be 156.7(36.2) and 19.9(5.2) amol.microgram-1 RNA, respectively. Parallel quantitative S1 nuclease protection assay yielded results of 131.2(64.2) and 9.4(5.2) amol.microgram-1 RNA. The cardiac CKB, but not CKM mRNA level, was twofold higher using the quantitative PCR method. However this discrepancy was abolished when compared to a higher stringency S1 nuclease protection assay. The cardiac CKB mRNA was 12.7% of the CKM level. This proportion remained the same from hearts with end stage cardiomyopathies of various aetiologies. CONCLUSIONS: This validated quantitative PCR method offers advantages over the S1 nuclease protection assay in that less RNA is required, the procedure is less dependent on RNA integrity and secondary structure, and multiple RNA species can be quantified simultaneously. The results also suggest that the abundance of the CKM and CKB mRNA level are coordinately regulated in the human heart.

Adolescent↗

Androgen receptor mRNA expression in Xenopus laevis CNS: sexual dimorphism and regulation in laryngeal motor nucleus.

Using Northern analysis, in situ hybridization, and nuclease protection assays, the expression and regulation of androgen receptor messenger RNA (AR mRNA) was examined in the CNS of juvenile Xenopus laevis. Only one of the AR mRNA isoforms expressed in X. laevis is transcribed in the CNS as shown by Northern blot analysis. Nuclease protection assays demonstrate that the expression of AR mRNA is higher in the brain stem than in the telencephalon and diencephalon. Although expression of AR mRNA is widespread throughout the CNS, cells of cranial nerve nucleus IX-X (N.IX-X) and spinal cord display the highest in situ hybridization signals in their cytoplasm. Double labeling using horseradish peroxidase and digoxigenin labeled AR probes reveals that laryngeal and anterior spinal cord motor neurons express AR mRNA. More cells express AR mRNA in N.IX-X of males than of females. The number of AR expressing cells in N. IX-X decreases following gonadectomy in both sexes, and dihydrotestosterone (DHT) treatment for 1 month reverses this effect. Increased expression of AR mRNA in the brain of DHT treated animals is also apparent in nuclease protection assays. Sex differences in number of AR expressing cells and hormone regulation of AR mRNA expression in motor nuclei may influence neuromuscular systems devoted to sexually differentiated behaviors.

Animals↗

Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia.

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, the reduced pteridine cofactor required for catecholamine (CA), indoleamine, and nitric oxide biosynthesis. We have used the reverse transcription-polymerase chain reaction technique, based on the published cDNA sequence for rat liver GTPCH, to clone a portion of the GTPCH transcript from rat adrenal gland mRNA and have used this clone for the analysis of GTPCH mRNA in brain and other tissues of the rat by northern blot, nuclease protection assay, and in situ hybridization. Two GTPCH mRNA transcripts of 1.2 and 3.8 kb in length were detected by northern blot, with the 1.2-kb form predominating in the liver and the 3.8-kb form in the pineal gland, adrenal gland, brainstem, and hypothalamic neurons maintained in culture. In situ hybridization studies localized GTPCH mRNA to CA-containing perikarya in the locus ceruleus, ventral tegmental area, and substantia nigra, pars compacta. Levels of GTPCH mRNA in central and peripheral catecholamine neurons determined by nuclease protection assay were increased twofold 24 h after a single injection of the CA-depleting drug reserpine; both the 1.2- and 3.8-kb transcripts were increased in the adrenal gland. Low levels of GTPCH mRNA were also detected by nuclease protection assay in the striatum, hippocampus, and cerebellum, brain regions that do not contain monoaminergic perikarya.

Animals↗

Interleukin-7 receptor expression and activation in nonhaematopoietic neoplastic cell lines.

The high-affinity human interleukin-7 (IL-7)R is a heterodimeric complex consisting of the IL-7Ralpha and common interleukin-2 receptor gamma (IL-2Rgamma(c)) chains. Activation of the IL-7R complex is associated with tyrosine and serine residue phosphorylation of a number of intracellular substrates leading to proliferation and induction of various cellular differentiation processes. In this study, we demonstrate, by S1 nuclease protection assay, immunoprecipitation and in vitro kinase assay that functional human (h) IL-7R is expressed in haematopoietic and nonhaematopoietic cell lines. The National Cancer Institute (NCI) tumour panel of 60 cell lines (NCI60) was screened for the expression of IL-7R mRNA by S1 nuclease protection assay, and IL-7R mRNA was detected in 9 of 12 leukemia, 3 of 7 lung, 4 of 6 CNS, 2 of 7 melanoma, 2 of 7 renal, 1 of 6 colon and 1 of 6 breast cancer cell lines. Immunoblot analysis of haematopoietic, lung cancer and brain tumour cell lines demonstrated expression of IL-7R, IL-2Rgamma(c) and p59 fyn, suggesting that the components of an IL-7R signalling network are present in nonhaematopoietic neoplastic cells. Immunoprecipitation of IL-7Ralpha followed by an in vitro kinase assay demonstrated functional receptor phosphorylation events in the lung cancer cells but not in the brain tumour cell lines. The expression of functional IL-7R on epithelial tumour cells may represent a potential target for receptor-directed therapy.

Brain Neoplasms↗

GABA transporter mRNA: in vitro expression and quantitation in neonatal rat and postmortem human brain.

A previously isolated rat cDNA clone encoding the membrane transporter for the neurotransmitter gamma-aminobutyric acid was expressed in transfected COS cells. The resultant transporter protein was characterized kinetically and pharmacologically. The apparent Kt (6.1 microM) and the pharmacological profile of a neuronal-type transporter observed in these mammalian cells were consistent with previous data obtained in Xenopus laevis oocytes. Post-natal levels of gamma-aminobutyric acid transporter mRNA in rat cerebellum, cerebral cortex and striatum (as measured by nuclease protection assay) transiently exceeded levels present in the adult brain. Human gamma-aminobutyric acid transporter mRNA also was measured by nuclease protection assay using as probe a human transporter cDNA homolog obtained by polymerase chain reaction. These studies suggest that quantitation of rat and human gamma-aminobutyric acid transporter mRNAs may provide a useful index of transporter gene expression.

Adult↗