定量测定黑鲷生长激素受体mRNA的液相杂交/RNase保护法

目的　建立液相杂交 核糖核酸酶保护测定 (RibonucleaseProtectionAssay,RPA)技术定量检测黑鲷(Sparusmacrocephalus)生长激素受体mRNA水平。方法　将黑鲷生长激素受体cDNA片断亚克隆至pGEM T载体 ,制备特异性的放射性反义RNA探针及正义RNA ,将反义RNA探针与正义RNA及样品总RNA进行液相杂交 ,用RNaseA和RNaseT1 降解杂交产物的单链RNA ,双链杂交体得到保护 ,然后检测杂交体的分子大小及放射强度。结果　检测出了黑鲷生长激素受体反义RNA探针与正义RNA及样品总RNA的特异性杂交片断 ,由此建立了定量测定黑鲷生长激素受体mRNA的液相杂交 RNase保护法 ,并采用该方法在黑鲷的多种组织中检测出了生长激素受体基因的表达 ,表达水平在肝脏中最高。该结果与我们曾采用生长激素放射受体分析法对黑鲷生长激素受体所研究的结果相吻合。结论　为进一步深入研究鱼类生长激素受体分子内分泌的调控理论提供了有力手段。     

A novel low molecular weight ribonucleic acid (RNA) related to transforming growth factor alpha messenger RNA

Human transforming growth factor alpha (TGF alpha) is coded for by an mRNA of about 4800 nucleotides. The cDNA sequence demonstrates that the 50 amino acid TGF alpha is embedded in a larger 160 amino acid precursor protein. We report here that in addition to the 4800 nucleotide TGF alpha mRNA, there is a novel second RNA species of about 350 nucleotides that hybridizes to a human TGF alpha cDNA probe. This small RNA species has been found in the RNA of several human tumor cells including HT1080, A549, A431, A2058, and A673. We have demonstrated an inverse relationship between the amounts of the 4800 nucleotide TGF alpha mRNA and the 350 nucleotide novel RNA in these human cells. Restriction enzyme cleavage of a human TGF alpha cDNA probe into three separate domains consisting of a processed coding region and 5'- and 3'-preprocessed coding and untranslated regions showed that only the 3'-untranslated region hybridized to the 350 nucleotide RNA. Using sense and anti-sense single-stranded 3'-untranslated region probes, we determined that the 350 nucleotide RNA band may be composed of multiple species of RNA which are related to the anti-sense DNA strand that is opposite to the strand that codes for the 4800 nucleotide TGF alpha mRNA.     

Quantitation of beta-tubulin mRNA in mouse brain by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. Two types of riboprobes for quantitating mouse beta-tubulin mRNA were prepared; one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with the mouse brain RNA, yielding heat-stable hybrids. The truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of mice of 10 and 50 days old were quantitated.     

Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.     

The expanding world of small RNAs in the hyperthermophilic archaeon Sulfolobus solfataricus

Archaeal L7Ae is a multifunctional protein that binds to a distinctive K-turn motif in RNA and is found as a component in the large subunit of the ribosome, and in ribose methylation and pseudouridylation guide RNP particles. A collection of L7Ae-associated small RNAs were isolated from Sulfolobus solfataricus cell extracts and used to construct a cDNA library; 45 distinct cDNA sequences were characterized and divided into six groups. Group 1 contained six RNAs that exhibited the features characteristic of the canonical C/D box archaeal sRNAs, two RNAs that were atypical C/D box sRNAs and one RNA representative of archaeal H/ACA sRNA family. Group 2 contained 13 sense strand RNA sequences that were encoded either within, or overlapping annotated open reading frames (ORFs). Group 3 contained three sequences form intergenic regions. Group 4 contained antisense sequences from within or overlapping sense strand ORFs or antisense sequences to C/D box sRNAs. More than two-thirds of these sequences possessed K-turn motifs. Group 5 contained two sequences corresponding to internal regions of 7S RNA. Group 6 consisted of 11 sequences that were fragments from the 5' or 3' ends of 16S and 23S ribosomal RNA and from seven different tRNAs. Our data suggest that S. solfataricus contains a plethora of small RNAs. Most of these are bound directly by the L7Ae protein; the others may well be part of larger, transiently stable RNP complexes that contain the L7Ae protein as core component.     

Characterization of specific cDNA background synthesis introduced by reverse transcription in RT-PCR assays

To block expression of NMDA receptor NR1 subunit, we injected into rat hippocampus a Herpes Simplex Virus type 1 derived vector bearing a sequence for NR1 antisense. RT-PCR assays with RNA from hippocampus of animals injected either with NR1 antisense vector, control vector or vehicle, showed an amplification signal compatible with NR1 antisense which could be attributed either to an endogenous NR1 antisense or to an artifact. RT-PCR was performed either with different primers or without primers in the RT, using RNA from different tissues. RNAse protection assay was carried out to characterize the amplified signal nature. Our results show that the template for the unexpected amplified fragment was NR1 mRNA currently expressed in nervous tissue. We considered this basal amplification of a mRNA in a RT-PCR assay as “background amplification”. After background subtraction, a significant signal only remained when samples from NR1 antisense vector injected animals were used, demonstrating that this was the only source for NR1 antisense. Background amplification at RT in the absence of primers, can constitute a troubling factor in quantitative nucleic acid determination, leading to major interference, particularly when both sense and antisense sequences are present in the sample. Since RT introduced a significant background signal for every gene analyzed, we propose that RT must be always performed also without primers. Then, this signal should be identified, quantified and subtracted from the specific reaction amplification signal.     

Improved silencing properties using small internally segmented interfering RNAs

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo.     

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc.     

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.     

Production of bulk amounts of universal RNA for DNA microarrays

In DNA microarray technology, repeatability and reliability are very important to compare multiple RNA samplesfrom different experiments. The application of common or universal RNA as a standard control equalizes the differences in hybridization parameters and array variations. For this purpose, high-quality reference RNA is necessary in bulk amounts. A novel approach was developed to get milligrams of sense or antisense RNA, starting from micrograms of pooled total RNA from different cell lines, tissues, or organisms. This method is inexpensive and allows further labeling procedures using poly(dT) or random oligomers as primers. In addition, amplified, sense reference RNA is suitable for standard labeling protocols, while the antisense reference RNA can be used with antisense RNA from the linear sample amplification method. Here we produced universal RNA for human, rat, and alfalfa and demonstrated the quality using specific cDNA microarrays.