A novel method for the isolation and study of a magnetotactic bacterium

The magnetococcus, a magnetotactic bacterium, has been grown in a complex simulated natural environment. Sufficiently pure samples of cells were obtained magnetically making axenic cultures unnecessary for many purposes. The magnetococcus is a Gram-negative coccus, 1.6 m in diameter and readily distinguished by highly refractile inclusions and its magnetotactic behavior. This organism is actively motile by means of two bundles of flagella. Electron dense ferromagnetic inclusions were localized between the flagellar bundles. Collections of magnetococci were morphologically homogeneous and negligibly contaminated by extraneous bacteria. DNA extracted from pooled collections of cells was homogeneous by analytical CsCl centrifugation. The guanine-cytosine content was 61.7%. Total iron by percent cellular dry weight was 3.8%. Comparisons with a previously described magnetotactic marine coccus were made.Non-Standard Abbreviations Tris Tris (hydroxymethyl) aminomethane buffer - EDTA Dipotassium ethylenediamine tetraacetic acid - GC Guanosine cytosine     

Highly insulin-responsive isolated rat heart muscle cells yielded by a modified isolation method

Freshly isolated adipocytes or cardiac myocytes appear to be subject to unspecific stimulation during isolation and subsequent handling, e.g. with respect to glucose transport. We have developed a modified procedure that yields rat cardiomyocytes with a very low basal, i.e. non stimulated hexose uptake rate (ca. 3 pmol * s-1 * mg protein-1 at 1 mM sugar), as compared to data reported by others. This low value correlates with the reported oxygen consumption of non-beating, isolated rat hearts, when these are perfused with glucose as the only substrate. The basal rate of glucose uptake in our quiescent cardiomyocytes is slightly lower than the value measured by others in beating rat hearts in vivo. Insulin (10 nM) stimulates 2-deoxy-D-glucose uptake 8- to 20-fold and 3-O-methyl-D-glucose uptake 14- to 20-fold, as compared to control. This insulin effect is markedly larger than that usually observed in isolated cardiomyocytes, but it is similar in magnitude to the stimulation of glucose transport reported for isolated, perfused rat hearts. In these cells, new stimulatory effects on the glucose transport, e.g. that of sulfhydryl reagents like phenylarsine oxide, become apparent. We conclude that the cardiomyocytes obtained by this modified method exhibit a basal glucose transport rate that is close to physiological values. These cells represent a new highly responsive model to detect and to investigate the effects of glucose transport stimulators (insulin, contraction etc.).     

A rapid high-yield isolation method for nuclear envelope ghosts

A method is presented for the isolation of nuclear envelopes from isolated Tetrahymena macronuclei. In principle, nuclei are treated with DNase and RNase at low Ca²⁺/Mg²⁺ concentration followed by an extraction with 1 NaCl. The major advantages of this method are: (1) Unfragmented nuclear envelopes are obtained in the form of ghosts consisting of two juxtaposing nuclear membranes interrupted by pores as revealed by thin-section and freeze-etch electron microscopy. (2) The ghosts are obtained in high yield (60%) within a short period (1 h). (3) The nuclear envelopes largely retain their lipid composition. An average ghost contains about 96% of total phospholipids of an average nucleus. Nuclei and ghosts reveal an almost identical pattern of phospholipids and fatty acids as shown by thin-layer and gas-chromatography. (4) The lipids in the ghosts largely remain arranged in bilayers as probed by electron spin resonance using 5-doxylstearic acid as a spin label.     

碱裂解提取质粒DNA的改进

碱裂解提取质粒DNA是分子生物学实验中常用的方法,但通常方法所提取的质粒往往含有大量的RNA和其他杂质.本文适时加入较高浓度的RNA酶和适当延长冰浴时间,结果得到了几乎没有RNA和其他杂质的高纯质粒DNA,不仅达到了分子生物学实验要求,而且可用作抗原检测抗dsDNA抗体.该法操作简单、经济、实用.     

Staphylococcal nuclease reviewed: A prototypic study in contemporary enzymology. I isolation; physical and enzymatic properties

Summary This is the first of a series of four articles in which the chemical, enzymological, and crystallographic work on Ribonucleate (deoxyribonucleate)-3-nucleotidohydrolase, EC 3.1.4.7, (Staphylococcal nuclease, Micrococcal nuclease) will be reviewed and correlated. This article discusses the purification of the enzyme and its general physical and enzymological properties. Subsequent articles will deal with specific studies of the nucleotide binding site, crystallographic studies of a nuclease-inhibitor complex, use of the nuclease as a model for protein folding and possible mechanisms for the action of the enzyme.This article is the first in a series of four parts. Part II will discuss chemical and physical studies related specifically to the nucleotide binding site. Part III will review the crystallographic studies as well as presenting a rational mechanism for the nuclease's mode of action. Part IV will deal with the use of the enzyme as a model for protein folding. Parts II–IV will appear in subsequent issues. The work from this laboratory has been supported by grants from the National Institute of General Medical Sciences, N. I. H. and the Robert A. Welch Foundation io F. A. COTTON and from the Welch Foundation to E. E. HAZEN, Jr.     

StreptoTag: a novel method for the isolation of RNA-binding proteins

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.     

Rapid isolation of monoclonal hybridoma cultures by a 'fusion-cloning' method: the requirement for aminopterin

Hybridomas were generated by fusing the Balb/c SP2/0 myeloma-like cell line with either: (i) splenocytes from Balb/c mice immunized with foot-and-mouth disease virus (FMDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), African swine fever virus (ASFV) or pig thymocytes; or (ii) lymph node cells from cattle immunized with FMDV. If the fusion mixtures were plated in cloning medium of methyl cellulose and HAT medium, small hybridoma colonies developed which rarely survived. Fusion mixtures were then plated in liquid HT medium on to 3T3/A31 feeder layers in 75 cm2 flasks, incubated at 37 degrees C for 24 h before adding aminopterin, and incubated for a further 2 to 4 days before cloning in methyl cellulose/HT medium. Without the aminopterin in the cloning medium, colonies of hybridomas, which could be cultured, developed from the majority of fusions. These colonies were isolated in HT medium over feeder layers and given two subcultures in HAT medium as a precaution against any reversion to aminopterin sensitivity during the cloning. No evidence of such reversions were seen, and recloning results suggested that the initial cloning was highly efficient at generating monoclonal cultures.     

An improved method for the quantitative isolation of rat liver nuclear RNA polymerases.

Rat liver nuclear RNA polymerases exist in two functional states, one of which is active towards the endogenous chromatin template (engaged enzyme), while the other is inactive (free enzyme) (Yu, F.L. (1974) Nature 251, 344-346). This paper reports the direct separation of these two populations of RNA polymerases from isolated rat liver nuclei by a simple extraction procedure. It is estimated that as much as 50% of the total nuclear RNA polymerase activity in normal rat liver may exist in the form of the free enzyme. Evidence is also presented to indicate that the free enzyme activity is easily lost when the nuclear isolation procedure involves the use of an isotonic buffer medium, or when the isolated nuclei are subjected to sonication as is required for the solubilization of the nuclear RNA polymerases by the conventional method. Based on these new findings, it is proposed that nuclei be isolated directly in hypertonic sucrose and that the free enzyme be extracted before the nuclei are subjected to sonication to solubilize the engaged enzyme. This method circumvents the loss of the free RNA polymerase population and, as a result, the total yield of the nuclear RNA polymerases is greatly increased. The possible functional role of the free RNA polymerase in gene expression is discussed.     

Detection and isolation of nuclear haplotypes by PCR-SSCP

SSCP (single-strand conformational polymorphism) is used widely in the field of human biomedicine, but its potential as a population genetics tool for the recovery of nuclear gene genealogies remains to be realized. We describe and illustrate a use for SSCP in the physical isolation of nuclear haplotypes that circumvents several difficulties associated with more conventional cloning procedures. The DNA sequence can be determined directly from the isolated haplotypes and used for phylogenetic inference. SSCP provides a convenient first step toward generating nuclear genealogies for population studies.     

Use of a modified cupric acetate method for the detection and quantitation of xylanolytic activities: a comparative study with the congo red method

A modified cupric acetate method for the screening and quantitation of xylanolytic activities was comparable with the more widely used congo red method with respect to sensitivity and ease of use and was shown to have points of merit over the latter. The use of a non-linear correction, in comparison to the conventional linear one, for the effect of dilution on the quantitative plate assay was evaluated.     

Corneal endothelium: a modified method for cultivation

A modified method for establishing cultures of rabbit corneal cells is described. The new technique utilized a Lucite disc in combination with a Tygon ring for growth of pure cell cultures and was compared with an explant method for growing cells. Each method provided adequate cell cultures for biochemical or ultrastructure studies of rabbit corneal cells, but the ring and disc method described here allowed the isolation of specific cell types without the interference of stromal cell contamination.     

Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method

Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources. J. Cell. Biochem. 71:363–374, 1998. © 1998 Wiley-Liss, Inc.     

A simple method for isolation of DNA fragments associated with the nuclear lamina in vivo

We describe a simple method for the purification of DNA fragments associated with the nuclear lamina in vivo. Ehrlich ascite tumor cells are first u.v.-irradiated to crosslink DNA to proteins. The nuclear lamina is then isolated and purified by low-speed centrifugation through a cushion of 40% sucrose. The material sedimenting through the created density barrier represents nuclear lamina of a very high purity, free from any DNA fragments except those which were in a crosslinking distance to it in vivo.