The viral RNA 3′- and 5′-end structure and mRNA transcription of infectious salmon anaemia virus resemble those of influenza viruses

The nucleotide sequences of the termini of two of the genomic segments of the negative strand RNA virus infectious salmon anaemia virus (ISAV) were determined. The sequence of the terminal 9 nucleotides at both ends of the viral RNAs was identical, and showed distinctive sequence homology with the conserved terminal sequences found in the orthomyxoviruses. For both ISAV genomic segments a computer-based secondary structure modelling indicated that the terminal 21-24 nucleotides were able to form self-complementary panhandle structures. Comparison with ISAV-derived mRNA sequences showed that ISAV mRNAs have heterogeneous 5'-ends, and are polyadenylated from a signal sequence 13-14 nucleotides downstream of the 5'-end terminus of the vRNA. Furthermore, the in vitro replication of ISAV was hindered by the RNA polymerase II inhibitor alpha-amanitin. These findings indicate that the mechanisms for replication of ISAV are similar to those of the orthomyxoviruses, and add to the previously reported structural similarities between ISAV and the orthomyxoviruses.     

3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

聚合酶3′外切活性对3′硫化修饰引物聚合反应的影响

目的 探讨硫化修饰的次3′末端不配对引物能否引发高保真DNA聚合酶介导的聚合反应的非成熟性终止,即所谓聚合反应的“关”效应。方法 采用配对及非末端不配对的3′硫化修饰引物,研究其对不同保真度DNA聚合酶引物延伸反应的影响。结果 非末端不配对的3′硫化修饰引物也能引发高保真DNA聚合酶介导的聚合反应非成熟性终止,而对低保真DNA聚合酶所介导的聚合反应则无明显影响。同时,3′硫化修饰的配对引物对不同保真度DNA聚合酶引物延伸反应均无影响。结论 硫化修饰的次3′末端不配对引物与3′末端不配对引物对引物延伸的影响相似,同样能引起高保真DNA聚合酶介导的聚合反应的“关”的效应。显然,在单基因遗传病的诊断及单核苷酸多态性(single nucleotide polymorphism,SHIP)的高通量分析等方面,硫化修饰的碱基特异性引物与高保真DNA聚合酶所构成的对SNP敏感的“开／关”系统,具有广阔的应用前景。     

利用3′-RACE法扩增到钙通道基因的3′-端片段

Ｃａ2 是细胞内的第二信使,涉及到细胞的信号传导、细胞运动、分裂、分化、神经递质的合成与释放,以及细胞凋亡等重要的基本生命过程。电压激活的钙离子通道在调节细胞内钙离子水平中起着至关重要的作用。以往报道表明,钙通道基因具有高度的多样性。已克隆的6个钙通道α1...     

Isolation of the missing 5′-end of the encoding region of the bovine leukemia virus cell receptor gene

Summary The missing 5-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5) was isolated from a lambda gt11 cDNA library using the³²P-labeledEcoRI-SamI fragment corresponding to the 5-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5 cDNA revealed that the 1058 bpEcoRI fragment at its 5-end contained a new 114 amino acid long sequence, and at its 3-end contained a completely identical 88 amino acid overlapping region with the 5-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.     

Mutants from V79 fibroblasts exhibiting hypersensitivity to aphidicolin and 3′-azido-3′-deoxythymidine

One variant, aph^hs-3 was previously isolated based on a hypersensitivity to nontoxic concentrations of aphidicolin, a specific inhibitor of DNA polymerases-alpha and delta. This variant was found to be more sensitive to temperatures above 35°C and to 10 µM of 3-azido-3-deoxythymidine (zidovudine, azidothymidine, or AZT) than the parental 743x cells. DNA polymerase activities in the cell extract or in the partially purified fraction by DEAE-cellulose (DE52) anion exchange column from aph^hs-3 were active at 40°C. No significant differences in deoxynucleoside triphosphate pools were observed at 34°C for both the parental 743x and aph^hs-3 cells. Revertants were isolated at 39°C: six revertants (aph^hs-3-tr1 through aph^hs-3-tr6) were obtained without aphidicolin; one revertant aph^hs-3-tar (the tar clone) was selected in aphidicolin (0.12 µM). The hypersensitivity to aphidicolin (Aph^hs) and AZT (AZT^hs) was cosegregated in the revertant aph^hs-3-tr5 (the tr5 clone), while the tar clone was not AZT^hs. There was a similar increase in the specific activity of³H-labeled DNA in all cell lines after additions of [³H]AZT or [³H]thymidine. Additions of purine or pyrimidine arabinosides (araT, araC, and araA) to all cell lines resulted in a similar cytotoxicity, suggesting the anabolism of dTTP was not defective in the tr5 clone. The spontaneous mutation rate at the hypoxanthine-guanine phosphoryltransferase locus using replating techniques and 6-thioguanine resistance selection was 5×10^–7, 2.2×10^–6, or 1.3×10^–6 per generation for the tr5, 743x, or tar cell lines, respectively. Most importantly, DNA polymerase activities in the cell extract of the revertant tr5 clone were inhibited by 0.5 µM AZTTP. In contrast, no inhibition was observed in those of the parental 743x and revertant tar cells. The cosegregation of both Aph^hs and AZT^hs in the tr5 revertant suggests that these two phenotypes may be a result of the same mutational event.     

3′碱基特异的PCR技术

引物3′末端的碱基处于一个对引物延伸至关重要的位置。如果该碱基与模板不互补(错配),就可能阻碍引物延伸,在PCR(聚合酶链反应)产物中没有特异长度扩增带。反之,如果该碱基与模板互补,则有特异长度扩增带。作者将引起β地中海贫血的三种最常见突变碱基设计并合成在引物3′末端上,根据PCR产物是否出现特异长度扩增带即可作出模板DNA有无相应突变的判断,而无须任何杂交或酶解。这种PCR称之为3′碱基特异的PCR(3′BS PCR)。     

5′和3′末端快速扩增法克隆多发性骨髓瘤负性相关基因DAZAP2

目的在多发性骨髓瘤患者中KIAA0058基因序列表达明显下调,从正常人骨髓单个核细胞中克隆该序列全长cDNA,以期更好地研究该序列结构,获得其功能信息。方法采用末端快速扩增法(RACE),进行多轮RT-PCR后,对PCR产物进行测序、拼接、证实,与NCBI核酸数据库进行比对。结果该序列全长cDNA长度为1913bp,登陆号为AY430097,与来源于人睾丸cDNA文库的序列DAZAP2同源性高达99%以上。该序列具有较短的5′非翻译区及长的3′非翻译区,开放阅读框序列从第84位碱基至590位碱基,具有poly(A)尾。结论利用RACE法,从骨髓单个核细胞中成功克隆了DAZAP2全长cDNA。     

Adenosine 3′∶5′-cyclic monophosphate mediates a 5-hydroxytryptamine-induced response in neonatal rat motoneurones

5-Hydroxytryptamine (5-HT) is present in nerve fibres descending from the brainstem Raphe nuclei to motoneurones and its release is thought to exert excitatory actions. 5-HT, applied from the outside, directly depolarizes spinal and cranial motoneurones in slices. This action of 5-HT is mediated, in part, by an inwardly rectifying cationic current, I _h. In cardiac cells, an equivalent current, i _f, has been shown to be directly activated by adenosine 35-cyclic monophosphate (cAMP) applied to the inside of the patch membrane. By applying the whole-cell method to thin slices of brainstem and spinal cord, we have shown that intracellularly applied cAMP and extracellularly applied dibutyryl cAMP or forskolin mimics the inward current induced by 5-HT. The selective cAMP phosphodiesterase inhibitor, Ro 20–1724, clearly prolonged the 5-HT-induced current. Maximal doses of dibutyryl cAMP or forskolin occluded the 5-HT-induced current. The broad spectrum protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methlypiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulphonamide (H-8) had no effect on the currents induced by 5-HT and forskolin. From these results, we suggest that activation of 5-HT receptors on the motoneuronal membrane stimulates formation of intracellular cAMP, thereby inducing the inward current, possibly by a direct action on I _h.     

The 3′terminal sequence of Chinese yam necrotic mosaic

Summary.  The nucleotide sequence of the 3′terminal 1,905 residues of the Chinese yam necrotic mosaic virus (ChYNMV) RNA genome was determined. It contains one long open reading frame, which consists of 1,671 nucleotides encoding a protein of 557 amino acid residues. A partial amino acid sequence of the coat protein determined from purified ChYNMV particles was identical to the portion of the amino acid sequence deduced from the determined nucleotide sequence, which suggests that the nucleotide sequence includes the coat protein gene. Surprisingly, a homology search using the deduced amino acids sequence of the coat protein revealed that ChYNMV is closely related to the genus Maclura-virus within the family Potyviridae, although the virus has long been considered to be a carlavirus. Identification of cylindrical cytoplasmic inclusions, which are characteristic of the family Potyviridae, in ChYNMV-infected Chinese yam cells, as well as the morphology and length (660 nm) of the purified virus particles, support including the virus as a tentative new member of the genus Macluravirus. Received September 1, 2000/Accepted March 17, 2001     

Measurement of the anti-HIV agent 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) by competitive ELISA

2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) is a thymidine analogue with potent anti-HIV activity in vitro and is currently being investigated therapeutically in patients with advanced HIV infection. We describe a first one-step competitive ELISA method developed for D4T measurement. Anti-D4T rabbit antibodies were raised against a D4T hemisuccinate-bovine serum albumin immunogen. A D4T-hemisuccinate-horseradish peroxidase conjugate and a monoclonal anti-rabbit IgG antibody insolubilized onto a microtiter plate were used as a tracer and capture system, respectively. The method was capable of detecting 2 ng/ml of D4T in cell cultures and 20 ng/ml of D4T in plasma samples previously separated in microconcentrator devices. Cross-reactivit y analysis showed that thymidine, D4T monophosphate, or azidothymidine, were weakly recognized by the ELISA and that thymine or other nucleosides were unreactive. The test was successfully used for the quantification of D4T in cell extracts from CEM or Molt 4 cell lines cultured with D4T and in the plasma of patients with advanced HIV infection, receiving D4T therapy. Moreover this ELISA could be used for the indirect quantification of D4T phosphorylated intracellular metabolites previously separated by reverse phase HPLC and hydrolyzed with alkaline phosphatase.     

真核生物mRNA3′非翻译区的调控功能

真核生物mRNA 3′非翻译区可以调节转录本的稳定性、亚细胞定位和翻译水平 ,决定某一特定mRNA的命运 ,是许多基因表达所必需的一个调节区。 3′非翻译区介导的功能的修饰可影响一个或多个基因的表达 ,从而导致疾病的发生。对相关mRNA 3′非翻译区的调节序列和与这些序列特异结合的蛋白质等具体信息的认识 ,将成为药物设计的新的分子靶。     

真核生物mRNA 3′非翻译区的调控功能

真核生物mRNA 3'非翻译区可以调节转录本的稳定性、亚细胞定位和翻译水平,决定某一特定mRNA的命运,是许多基因表达所必需的一个调节区.3'非翻译区介导的功能的修饰可影响一个或多个基因的表达,从而导致疾病的发生.对相关mRNA 3'非翻译区的调节序列和与这些序列特异结合的蛋白质等具体信息的认识,将成为药物设计的新的分子靶.     

The Y3** ncRNA promotes the 3′ end processing of histone mRNAs

We demonstrate that the Y3/Y3** noncoding RNAs (ncRNAs) bind to the CPSF (cleavage and polyadenylation specificity factor) and that Y3** associates with the 3′ untranslated region (UTR) of histone pre-mRNAs. The depletion of Y3** impairs the 3′ end processing of histone pre-mRNAs as well as the formation and protein dynamics of histone locus bodies (HLBs), the site of histone mRNA synthesis and processing. HLB morphology is also disturbed by knockdown of the CPSF but not the U7-snRNP components. In conclusion, we propose that the Y3** ncRNA promotes the 3′ end processing of histone pre-mRNAs by enhancing the recruitment of the CPSF to histone pre-mRNAs at HLBs.     

Suppression of lymphocyte adenosine 3′ : 5′-cyclic monophosphate (cAMP) by delta-9-tetrahydrocannabinol

Delta-9-tetrahydrocannabinol (THC) is the major psychoactive component of marijuana. Suppression of mitogen-stimulated blastogenesis of human lymphocytes in vitro by THC was previously demonstrated. This effect was shown to be concentration dependent with the non-toxic concentrations 5, 7.5, and 10 μg THC/ml showing the greatest suppression. However, the mechanism(s) by which THC induces suppression are still unclear. The current study examines the effect of THC on the adenosine 3′ : 5′-cyclic monophosphate (cAMP) pathway second messenger system, which is involved in activation of human peripheral blood lymphocytes. Lymphocyte cAMP levels were stimulated using three hormone receptor stimulators, isoproterenol, histamine, or 5′-N-ethylcarboxamide adenosine (NECA), each of which utilizes a different receptor to enhance cAMP production. THC suppressed cAMP levels independently of the hormone and receptor utilized. Levels of cAMP in non-mitogen-stimulated peripheral blood mononuclear cells and plastic non-adherent lymphocytes, as well as cells stimulated with phytohemmagglutinin, were suppressed by THC. Suppression of cAMP production by THC was further examined to determine whether inhibition involved a GTP-binding protein (G_i), which is known to down-regulate cAMP production. Cells were pre-treated with pertussis toxin to inhibit G_i activity; this blocked the THC-induced suppression of cAMP production. These results suggest that THC can exert its effects on second messenger systems at the lymphocyte membrane level, and that a pertussis toxin-sensitive G_i protein may be involved. Thus, second messenger regulated pathways may be involved in THC-induced immune suppression. However, the relationship between alteration of cAMP production and suppression of lymphocyte function due to the presence of THC in the medium remains to be established.     

Rice tungro spherical virus: Nucleotide sequence of the 3′ genomic half and studies on the two small 3′ open reading frames

Rice tungro spherical virus (RTSV) consists of a single-stranded RNA genome of about 12 kilobases that contains one large open reading frame, ORF 1 and two small ORFs 2 and 3 at its 3 end (Shen et al., 1993, Virology 193:621–630); it was suggested that ORF 2 was expressed via a frameshift. To study the genomic information of RTSV and the variation between different RTSV isolates, the 3 half of a Philippine isolate and parts of a Thai and an Indian isolate were cloned and sequenced. Significant sequence differences were found in ORF 2 and in the 3 non-translated region. Additional stop codons have been revealed in the previously described ORF 2 in several independent clones from the three different virus isolates, the most conserved stop codon in the middle of ORF 2 being confirmed by direct RNA sequencing. These results suggest that ORF 2 could only express a peptide of about 5 kDa instead of 12 kDa as proposed earlier. Polyclonal antisera were raised against ORF 2 and 3 proteins as fusions with glutathione-S-transferase. Using these antisera we failed to detect any virus-specific peptides in extracts from infected rice plants and in virus preparations. The nucleotide sequence of the 3 end of our RTSV isolates contains several small ORFs and does not contain a repeat of 256 nucleotides found in the published sequence. These results indicate that RTSV could contain an unusually long 3 non-coding region of 1240 nucleotides in length.