A fluorescence-labeling method for sequencing small RNA on polyacrylamide gel.

A practical fluorescence-labeling method for sequencing small RNAs by the traditional 'direct read out' on polyacrylamide gel electrophoresis was established. The 3' terminus of RNA was oxidized into dialdehyde by sodium periodate and then labeled with fluorescein-5-thiosemicarbazide through the condensation reaction between carbazide and aldehyde. The fluorescence-labeled RNA was partially degraded enzymatically and fractionated by polyacrylamide gel electrophoresis. The fluorescent bands were visualised by ultraviolet photography. A partial sequence of yeast 5S rRNA was determined. The result indicates that this method can be used in sequencing small RNAs rapidly, conveniently and safely.     

快速提取、纯化叶绿体4.5SrRNA的方法

我们采用植物叶与热缓冲液、苯酚直接混合(约65℃)匀浆,离心抽提和乙醇沉淀后,得到植物叶总RNA。经聚丙烯酰胺凝胶电泳分离、纯化,即可得到叶绿体4.5S rRNA,此法不仅操作简单,而且得率高。 同时,经过对同一植物的不同组织或不同细胞组分,如根、细胞质、叶绿体和叶绿体核糖体小分子RNA的提取与鉴定,以简便的方法证明了4.5S rRNA是叶绿体核糖体成份,也证明了我们所采用的提取、纯化4.5SrRNA方法的可靠性。     

Effect of alpha-actinin on actin structure. Actin ATPase activity

The accumulation of low molecular weight RNAs in Escherichia coli cells following amino acid or energy source starvation was examined using two-dimensional polyacrylamide gel electrophoresis. 32P-labeled small RNA prepared from serine- or isoleucine-starved stringent strain (relA+) cells was shown to display gel patterns that were grossly different from that of unstarved cells. It appears that the deprivation of serine or isoleucine has little or no inhibitory effect on the accumulation of transfer RNA cognate to the deprived amino acid. This is demonstrated by a relative increase in the concentrations of small RNAs that can be charged with serine or isoleucine following starvation of these amino acids. However, small RNAs labeled during starvation of phenylalanine or energy source showed gel patterns similar to that of control cells. This suggested a heterogenous response in the accumulation of some low molecular weight RNAs, presumably transfer RNAs, following starvation of different amino acids.     

Low-Molecular-Weight RNAs of Murine Sarcoma Virus: Comparative Studies of Free and 70S RNA-Associated Components

Several low-molecular-weight RNAs were released from mouse leukemia sarcoma virus 70S RNA under conditions of thermal denaturation. One of them, the 70S-associated 8S RNA, exhibits a number of structural properties characteristic of a previously described free viral 8S RNA. This similarity was revealed by polyacrylamide gel electrophoresis and nucleotide composition.     

大鼠4.5S RNAs的cDNA克隆与序列分析

利用ＲＴ－ＰＣＲ方法,首次从大鼠肝脏细胞总ＲＮＡ中扩增出４．５Ｓ ＲＮＡｓ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,经酶切电泳鉴定,然后测序。与报道的小鼠和仓鼠４．５Ｓ ＲＮＡｓ序列进行了比较研究,并对该分子的结构特点进行了初步分析。     

A new series of RNAs associated with the genome of spleen focus forming virus (SFFV) and poly(A)-containing RNA from SFFV-infected cells.

A series of low molecular weight RNAs (4.5 to 5.5S) as well as other 4 to 7S RNAs were dissociated from genomic RNA of spleen focus forming virus (SFFV) by heating. On two dimensional polyacrylamide gel electrophoresis, this series of RNAs gave a series of more than thirty spots. RNase T1 fingerprints of these spots were identical except for differences in 3'-terminal oligonucleotides, which were mainly due to different numbers of uridylic acid residues, larger RNA-molecules containing poly(U)sequences at their 3'-termini. This series of RNAs is also associated with poly(A)-containing nuclear and cytoplasmic RNAs from SFFV-infected cells.     

Recent studies of some RNAs and proteins in amoebae

Progress on various aspects of nucleic acids and protein synthesis in amoebae has been reviewed. The RNA molecules involved in the character changes seen after micro-injection of non-homologous cytoplasmic fractions have been isolated after polyacrylamide gel electrophoresis, and their approximate molecular weights calculated. Injection of these RNA molecules was shown to alter the response of recipient cells to growth in streptomycin and neomycin.The relative molecular weights of cytoplasmic ribosomal RNAs have been estimated using both aqueous and formamide gel electrophoresis. Some attempts to characterize the nuclear RNAs seen on aqueous polyacrylamide gels, and to evaluate this data with that published by other workers have been made. Results from assays of DNA- and RNA-directed DNA polymerase activity are considered in relation to those from other eukaryotes.Problems arising after attempts to use rabbit globin messenger RNA to direct globin synthesis in amoebae, and the possibilities of using minature gel systems and small cell numbers to identify proteins and RNAs after various experimental treatments are discussed.     

Separation and purification of small quantitites of specific RNA species by polyacrylamide gel electrophoresis using prestained RNAs as markers.

A method is described for the separation and purification of different molecular species of RNA in microquantities using polyacrylamide gel electrophoresis. The experimental procedure consists of the following steps; (i) partial prestaining of RNA with methylene blue at a concentration of the dye which would not affect the electrophoretic migration of the RNA bands, but which would permit visual observation of the migrating bands during electrophoresis; (ii) use of short columns just sufficient to achieve separation of each species of RNA as a single compact band rather than a series of bands; (iii) trapping of each of the eluting RNA species from the gel on DEAE-cellulose dises in sequential order; and (iv) elution of the RNAs from the DEAE-discs by extraction with triethylammonium bicarbonate and recovery of the corresponding RNAs by lyophylization.     

Enhanced resolution of circular and linear molecular forms of viroid and viroid-like RNA by electrophoresis in a discontinuous-pH system

A discontinuous-pH polyacrylamide gel electrophoresis system is described. An increase in the pH differential between the gel and the running buffer enhances the separation of low molecular weight circular and linear RNA molecules. Highly purified preparations of the circular form of viroids can be obtained with this procedure. Since all the linear RNAs of similar molecular weight migrate with the front, a relatively clean background can be obtained even when crude extracts are used. This facilitates an improved separation and identification of similarly sized viroid-like RNAs. The conditions of electrophoresis in low salt and 8 M urea also permit the effective transfer of RNA molecules directly to nylon-based membranes without any additional denaturation treatment.     

Separation of t-RNAs by two-dimensional polyacrylamide gel electrophoresis

A pH 5.8 polyacrylamide gel electrophoresis buffer is described. Electrophoresis in this MES-citrate system at pH 5.8 separates E. coli transfer RNAs into 15 bands using 15% acrylamide gels. Polyacrylamide gel electrophoresis in a second dimension at pH 8.3 further resolves E. coli t-RNAs into 20 spots.     

北京鸭珠蛋白mRNA的分离纯化及其cDNA的合成

 从人工贫血的北京鸭网织红细胞中直接提取总RNA,经Oligo(dT)-纤维素柱层析分离获得珠蛋白mRNA,并经蔗糖密度梯度离心首次得到了电泳单一条带的北京鸭球蛋白mRNA。从凝胶电泳以及蔗糖密度梯度离心鉴定其沉降系数为9S。在麦胚无细胞体外翻译体系中测定了它们的蛋白翻译活力。鸭珠蛋白mRNA促进了~3H-亮氨酸参入新生蛋白的活力,达到对照组的10倍。所翻译的蛋白产物在SDS-聚丙烯酰胺凝胶上的电泳行为与天然鸭珠蛋白一致。 经Oligo(dT)-纤维素及蔗糖密度梯度离心提纯的珠蛋白mRNA,在AMV反转录酶及DNA聚合酶的作用下,分别合成了单链及双链cDNA。其双链链长,经凝胶电泳分析,约为500碱基对。     

枯草芽孢杆菌内切木聚糖酶的分离纯化与鉴定

目的：建立一种简便、快速的木聚糖酶分离和提取方法。方法：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,分离纯化枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中的木聚糖酶,进一步用薄层色谱和高压液相色谱对木聚糖酶进行鉴定。结果：采用活性聚丙烯酰胺凝胶电泳和均质提取法相结合,从枯草芽孢杆菌(Bacillus subtilis)固体培养基发酵产物中分离得到了两种内切木聚糖酶,酶解桦木木聚糖的产要产物以木二糖和木三糖为主。结论：活性聚丙烯酰胺凝胶电泳和均质提取法相结合是一种新的分离纯化木聚糖酶的简便、有效方法。     

Low-molecular-weight RNAs of Moloney murine leukemia virus: identification of the primer for RNA-directed DNA synthesis.

The small RNAs of Moloney murine leukemia virus (M-MuLV) were fractionated into at least 15 species by two-dimensional polyacrylamide gel electrophoresis. The pattern of small RNAs is significantly different from that of Rous sarcoma virus. A subset of the virion small RNAs is associated with the genome RNA in the 70S complex. One of the associated molecules, a cellular tRNA, is tightly bound to the genome RNA and serves as the major primer for M-MuLV RNA-directed DNA synthesis in vitro.     

Chemical and physical properties of mammalian mitochondrial aminoacyl-transfer RNAs. I. Molecular weights of mitochondrial leucyl- and methionyl-transfer RNAs.

The sedimentation and electrophoretic properties of Syrian hamster cytosolix and mitochondrial methionyl- and leucyl- +RNAs have been compared under denaturing conditions. Mitochondrial leucyl-tRNA could be separated into three species by chromatography on RPC-5. Their apparent molecule weights as determined by polyacrylamide slab gel elecltrophoresis were 23 000 for one species and 24 000 for the other two compared to the five cytosolic leucyl-tRNA species whose apparent molecular weights ranged from 26 000 to 28 000. Mitochondrial leucyl-tRNAs sedimented more slowly than their cytosolic counterparts, again indicating a lower molecular weight. The apparent molecular weights of the mitochondrial methionyl-tRNAs were identical or only slightly lower than their cytosolic counterparts as determined by polyacrylamide slab gel electrophoresis but both mitochondrial methionyl-tRNA and formylmethionyl-tRNA sedimented slightly more slowly than cytolsolic methionyl-tRNA. It is suggested that mitochondrial tRNAs fall into the size range of other t RNAs and might be uniform in size.     

Fluorography of tritium-labelled proteins in immunoelectrophoresis

A method is described for detecting 3H-labelled proteins in immunoelectrophoretic systems performed on agarose gels. The method is based on the incorporation of a polyacrylamide gel into the agarose gel after the electrophoresis. This mixed gel has the characteristics of a polyacrylamide gel, making it possible to use fluorography as has been described for polyacrylamide gels. The applicability of the fluorography method is demonstrated by analyzing 3H-labelled human serum albumin and 3H-labelled pig intestinal brush border proteins by quantitative immunoelectrophoresis.     

Isolation and characterization of casein mRNAs from lactating ewe mammary glands.

RNA from bound polysomers of lactating ewe's mammary gland directs the synthesis of the three major milk proteins (alphas, beta and kappa-caseins) in a cell-free system derived from rabbit reticuyocytes. The "in vitro" product was identified by immunoprecipitation with specific antibodies and by electrophoresis in SDS polyacrylamide gel. Each of these messengers was purified from 20 to 25 fold from total membrane-bound polysomal RNA using poly U-Sepharose chromatography. This purified fraction assayed in a reticulocyte cell-free system is able to direct also the synthesis of 2 minor secretory proteins (beta-lactoglobulin and alpha-lactalbumin). The messenger RNAs purified by hybridization to poly U-Sepharose have a sedimentation coefficient of about 12 S and an apparent molecular weight of approximatively 3.5 s 10-5 daltons was observed by polyacrylamide gel electrophoresis under denaturing contitions. This value which correspond to about 900 nucleotides is significantly greater than the number expected for coding milk proteins.     

Electrophoretic analysis of the molecular weight of murine mammary tumor virus RNA.

Molecular weight determinations of native and subunit RNAs of murine mammary tumor virus (MuMTV), a type B oncornavirus, were performed by polyacrylamide gel electrophoresis and compared with molecular weights of well-characterized avian cellular RNAs and tobacco mosaic virus RNA. From extrapolations of semilog plots of the molecular weights of the standard RNAs versus relative electrophoretic mobilities and Ferguson plots, the subunit and native RNAs of MuMTV were found to possess molecular weights of 2.93 X 10(6) and 6.45 X 10(6), respectively. These data support the assumption that two subunit molecules comprise the native RNA of MuMTV.