Rapid alkaline transfer of low molecular weight DNA from NuSieve GTG agarose gels

The rapid alkaline transfer of high molecular weight DNA from agarose gels to nylon membranes has greatly decreased the time required for setup of Southern transfers. This technique has been used to resolve genomic DNA greater than 1000 base pairs by conventional electrophoresis on 1% agarose gels followed by alkaline transfer to nylon membrane. Now we report that this rapid alkaline method can be used for the transfer of low molecular weight DNA fragments (10 to 1000 base pairs) from NuSieve GTG agarose gels to nylon membrane.     

Denaturing RNA electrophoresis in TAE agarose gels

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris-acetate-ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-(N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.     

A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels.

Defined RNA fractions can be recovered from low gelling temperature agarose gels by a combination of agarose melting at 65°C and phenol extraction. By this approach RNA molecules up to a size of 37 kb can be eluted undegraded with a recovery of 60–90%. The method is applicable also to DNA and the eluted DNA can be correctly cleaved with restriction endonucleases as shown for λDNA using EcoRI.     

The electrophoretic mobility of double-stranded RNA in polyacrylamide gels as a function of molecular weight.

A re-evaluation of the mobility of double-stranded RNA on polyacrylamide gels over a molecular weight range of 0.46-6.3 . 10(6) was carried out using double-stranded RNAs of: bacteriophage ?6; virus like particles or mycoviruses of Penicillium chyrsogenum, Penicillium stoloniferum and Helminthosporium maydis, and reovirus type III. When the relative mobility on polyacrylamide gels was plotted as a function of log molecular weight, a smooth curve could be drawn which passed through all points. The implications of these findings to the determination of molecular weight of double-stranded RNA by polyacrylamide gel electrophoresis are discussed.     

Identification of bacteria by low molecular weight RNA profiles: a new chemotaxonomic approach

A new chemotaxonomic method is presented for the identification of eubacteria. This method is based on one-dimensinal gel electrophoresis of total RNA extracts from eubacteria. Only low molecular weight (<150 nucleotides) RNA, comprising 5S ribosomal and transfer RNA, was used for the identification. The high resolution of the electrophoresis, better than half a nucleotide, allowed construction of low molecular weight (LMW) RNA profiles that contained 10 to 20 bands per strain. LMW RNA profiles of a set of eubacterial reference strains showed on variation in dependence on culture conditions or physiological state of the cells. Computer-assisted data evaluation, including six molecular weight markers, enabled the calculation of relative nucleotide units (RNU) for every band. The resulting normalized band pattern allowed the identification of identical strains on different gels. The relative position of the single bands from the different groups of RNAs made an identification of bacterial strains to genus and often species level possible.Especially valuable for the identification were the large, class 2 tRNAs that showed certain variation among species of the same genus and varied considerably among different genera. RNA profiles can provide a rapid and inexpensive screening technique for the taxonomic classification of single bacterial strains. Potential fields of application for this technique might be bacterial taxonomy, biotechnology and ecology.     

Separation of long RNA by agarose–formaldehyde gel electrophoresis

We describe a method to facilitate electrophoretic separation of high-molecular-weight RNA species, such as ribosomal RNAs and their precursors, on agarose–formaldehyde gels. Two alternative “pK-matched” buffer systems were substituted for the traditionally used Mops-based conductive medium. The key advantages include shortened run times, a 5-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a streamlined work flow that helps to minimize errors and is broadly applicable to agarose gel electrophoresis of RNA samples and their subsequent analysis by Northern blotting.     

Separation and purification of high molecular weight glycoproteins using agarose gel electrophoresis.

Several components of the extracellular matrix in the molecular weight range of 220 kDa to 150 kDa were purified by preparative electrophoresis on 2.5% Pro-Sieve agarose gels. These high molecular weight glycoproteins, separated under reducing conditions, were recovered in solution by extraction of individual agarose gel slices and analyzed on sodium dodecyl sulfate polyacrylamide gels and Western blots. This simple method permitted the separation and recovery of the laminin B chains (220 kDa and 205 kDa) and entactin (150 kDa) and may prove useful for the purification of other high molecular weight species.     

Flexibility difference between double-stranded RNA and DNA as revealed by gel electrophoresis

A systematic study of agarose gel electrophoresis of double-stranded RNA in the kilobase range of sizes was performed. The dsRNA to dsDNA relative mobility was found to depend on gel concentration: in low density gels RNA moves slower and in high density gels - faster than DNA of the same molecular size. The electrophoretic differences were interpreted within the reptation theory to be mainly due to the molecular stiffness differences. The dsRNA persistence length was roughly estimated to be about twice as great as that of DNA.     

Low molecular weight RNA species from chromatin.

Several methods of preparing low molecular weight RNA from chick embryo chromatin have been examined. Traditional methods for dissociating chromatin utilizing high concentrations of salt (greater than 2 M) followed by high-speed centrifugation resulted in very low yields of RNA. Increased yields of RNA were obtained by treating chromatin at lower salt concentration (0.2-0.5 M). By using low salt extraction and sodium dodecyl sulfate-phenol deproteinization, six to eight low molecular weight homogeneous RNA species were isolated from chick embryo chromatin and mouse myeloma chromatin. In the myeloma system, all these RNAs are metabolically stable. Each component is homogeneous as examined by gel electrophoresis and hybridizes with mouse DNA at a rate consistent with a single species. There are multiple gene copies for these RNA species in the mouse genome, varying from 100 to 2000 copies for the different species. One of these RNAs is identical with 5S rRNA. In addition, the redundancy of genes for 18S, 28S, and 5S rRNA and tRNA was determined. Approximately 300 copies for 18 and 28S rTRNA and 500 copies for 5S rRNA were found. tRNAs were on an average 110-fold redundant with about 55 different species measured.     

Intracellular distribution of low molecular weight RNA in Chironomus tentans

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4–5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10⁵ and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10⁵ and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10⁴ and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (β-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4–5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Isolation of low molecular weight nuclear RNA from small numbers of nuclei with cetyltrimethylammonium bromide

A method is presented for the isolation of low molecular weight nuclear (LMN) RNAs from small numbers of nuclei. Cetyltrimethylammonium bromide (CTAB) is used to precipitate small quantities of whole nuclear RNA from dilute aqueous solution following phenol-SDS extraction of purified nuclei. No carrier RNA is necessary during the precipitation step. LMN RNAs are separated from whole nuclear RNA by electrophoresis on polyacrylamide gels. No further purification of the RNA is necessary prior to electrophoresis. Both radioactivity and absorbance profiles of the LMN RNAs on the gels can be obtained. Thus, specific activities of labeled LMN RNA species can be estimated.     

Physical and chemical characterization of purified ovalbumin messenger RNA.

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.     

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA.Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to α-amanitin. In isolated nuclei, both RNA species are labeled with β-³²P-labeled GTP, which suggests that they are initiated at separate promoter sites.     

5S RNA AND TRANSFER RNA SYNTHESIS IN GERMINAL VESICLE OOCYTES AND DURING HORMONALLY-INDUCED MEIOTIC MATURATION OF STARFISH OOCYTES

The incorporation of ³H-guanosine as ³H-GMP into 5S RNA and into transfer RNA (tRNA) was examined in isolated large germinal vesicle oocytes, in isolated mature ootids and during and subsequent to hormonally (l-methyladenine)-induced meiotic maturation in the starfish, Asterias forbesii .Purified soluble RNA ¹ preparations at each stage were fractionated by electrophoresis on 10% polyacrylamide gels, while high molecular weight RNAs were resolved by subjecting total RNA samples to electrophoresis on 2.4% acrylamide+0.5% agarose gels. The results showed that large germinal vesicle oocytes, containing a single compact nucleolus, synthesize 5S RNA and tRNA as well as the previously-reported (1, 23-26) nucleolar rRNAs. In contrast, during and subsequent to hormonally-induced meiotic maturation, after germinal vesicle braekdown and nucleolar dissolution, the synthesis of 5S RNA and tRNA continues in the absence of detectable high molecular weight rRNA synthesis.     

Low molecular weight RNAs hydrogen-bonded to nuclear and cytoplasmic poly(a)-terminated RNA from cultured chinese hamster ovary cells

A group of RNAs 90–100 nucleotides long were isolated by melting them from poly(A)-terminated nuclear or cytoplasmic RNA from cultured Chinese hamster ovary cells. Conditions that favor hydrogen bond formation allowed the reassociation of these low molecular weight RNAs with poly(A)-terminated RNA. The nuclear poly(A)-terminated molecules contained 1.3 moles of the low molecular weight RNAs per mole of poly(A), while the cytoplasmic poly(A)-terminated RNA contained only one seventh as much. These low molecular weight RNAs were also isolated from the total 4S RNA of either the nucleus or cytoplasm by polyacrylamide gel electrophoresis. They formed a prominantly labeled band of RNA in the gels after cells had been labeled with H₃³²PO₄ for 4 hr. The low molecular weight RNAs melted from the nuclear poly(A)-terminated RNA were slightly different (although not necessarily in primary nucleotide sequence) from those melted from the cytoplasmic poly(A)-terminated RNA.