A simple and effective separation and purification procedure for DNA fragments using Dodecyltrimethylammonium bromide

In this work we describe a simple two step separation procedure for the separation and purification of short DNA fragments. The first step involves precipitating the DNA using the cationic surfactant dodecyltrimethylammonium bromide. Dodecyltrimethylammonium bromide, unlike cetyltrimethylammonium bromide will not precipitate DNA before complexation is complete thus providing a high purity DNA. The second step involves dissolution of the DNA-dodecyltrimethylammonium complex in 75% ethanol, followed by precipitation of the Sodium-DNA salt, by titrating in a salt solution. This method is particularly suited to purification of short fragments as it does not require high salt concentrations in the ethanol precipitation step, which can be damaging for short DNA. The ability of dodecyltrimethylammonium bromide to remove ethidium bromide from intercalation sites on the DNA is also discussed     

Genetic dissection of collagen-induced arthritis in Chromosome 10 quantitative trait locus speed congenic rats: evidence for more than one regulatory locus and sex influences

Rat Chromosome 10 (RNO10) harbors Cia5, a non-MHC quantitative trait locus (QTL) that regulates the severity of type II collagen-induced arthritis (CIA) in DAxF344 and DAxBN F2 rats. CIA is an animal model with many features that resemble rheumatoid arthritis. To facilitate analysis of Cia5 independently of the other CIA regulatory loci on other chromosomes, DA recombinant QTL speed congenic rats, DA.F344(Cia5), were generated. These QTL congenic rats have a large chromosomal segment containing Cia5 (interval size < or =80.1 cM) from CIA-resistant F344 rats introgressed into their genome. Phenotypic analyses of these rats for susceptibility and severity of CIA confirmed that Cia5 is an important disease-modifying locus. CIA severity was significantly lower in the Cia5 congenic rats than in DA controls. We also generated DA Cia5 speed sub-congenic rats, DA.F344(Cia5a), which had a smaller segment of the F344 genome, Cia5a, comprising only the distal q-telomeric end (interval size < or = 22.5 cM) of Cia5, introgressed into their genome. DA.F344(Cia5a) sub-congenic rats also exhibited reduced CIA disease severity compared with the parental DA rats. The regulatory effects in both congenic strains were sex influenced. The disease-ameliorating effect of the larger fragment, Cia5, was greater in males than in females, but the effect of the smaller fragment, Cia5a, was greater in females. We also present an improved genetic linkage map covering the Cia5/Cia5a region, which we have integrated with two rat radiation hybrid maps. Comparative homology analysis of this genomic region with mouse and human chromosomes was also undertaken. Regulatory loci for multiple autoimmune/inflammatory diseases in rats (RNO10), mice (MMU11), and humans (HSA17 and HSA5q23-q31) map to chromosomal segments homologous to Cia5 and Cia5a.     

Identification of a signal peptide for oryzacystatin-I

 A previously unidentified extension of an open reading frame from the genomic DNA of Japonica rice (Oryza sativa L.) encoding oryzacystatin-I (OC-I; access. M29259, protein ID AAA33912.1) has been identified as a 5′ gene segment coding for the OC-I signal peptide. The signal peptide appears to direct a pre-protein (SPOC-I; Accession No. AF164378) to the endoplasmic reticulum, where it is processed into the mature form of OC-I. The start codon of SPOC-I begins 114 bp upstream from that previously published for OC-I. A putative proteolytic site, which may yield a mature OC-I approximately 12 residues larger than previously described, has been identified within SPOC-I between Ala-26 and Glu-27. The signal peptide sequence was amplified by polymerase chain reaction using genomic DNA from O. sativa seedlings and ligated to the 5′ end of the truncated OC-I gene at the endogenous SalI site. Partially purified protein extracts from Escherichia coli expressing SPOC-I reacted with polyclonal antibodies raised against OC-I and revealed a protein of the expected molecular weight (15,355 Da). In-vitro translation of SPOC-I in the presence of microsomal membranes yielded a processed product approximately 2.7 kDa smaller than the pre-protein. Nicotiana tabacum L. cv. Xanthi plants independently transformed with the SPOC-I gene processed SPOC-I and accumulated the mature form of OC-I (approximately 12.6 kDa), which co-migrated with natural, mature OC-I extracted from rice seed when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Received: 29 July 1999 / Accepted: 25 August 1999     

The Atrazine Catabolism Genes atzABC Are Widespread and Highly Conserved

Pseudomonas strain ADP metabolizes the herbicide atrazine via three enzymatic steps, encoded by the genes atzABC, to yield cyanuric acid, a nitrogen source for many bacteria. Here, we show that five geographically distinct atrazine-degrading bacteria contain genes homologous to atzA, -B, and -C. The sequence identities of the atz genes from different atrazine-degrading bacteria were greater than 99% in all pairwise comparisons. This differs from bacterial genes involved in the catabolism of other chlorinated compounds, for which the average sequence identity in pairwise comparisons of the known members of a class ranged from 25 to 56%. Our results indicate that globally distributed atrazine-catabolic genes are highly conserved in diverse genera of bacteria.Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)- 1,3,5-triazine] is a herbicide used for controlling broad-leaf and grassy weeds and is relatively persistent in soils (51). Atrazine and other s-triazine compounds have been detected in ground and surface waters at levels exceeding the Environmental Protection Agency’s maximum contaminant level of 3 ppb (30).Microbial populations exposed to synthetic chlorinated compounds, such as atrazine, often respond by producing enzymes that degrade these molecules. Most of our current understanding of the genes and enzymes involved in atrazine degradation derives from studies using Pseudomonas strain ADP, in which the first three enzymatic steps in atrazine degradation have been defined (6, 14, 15, 48). The genes atz A, -B, and -C, which encode these enzymes, have been cloned and sequenced. Atrazine chlorohydrolase (AtzA), hydroxyatrazine ethylaminohydrolase (AtzB), and N-isopropylammelide isopropylaminohydrolase (AtzC) sequentially convert atrazine to cyanuric acid (6, 14, 15, 48) (Fig. ​(Fig.1).1). Cyanuric acid and related compounds are catabolized by many soil bacteria (10, 11, 17, 24, 26, 61), and by Pseudomonas sp. ADP, to carbon dioxide and ammonia (35). This provides the evolutionary pressure for the atzA, -B, and -C genes to permit bacterial growth on the more than one billion pounds of atrazine that have been applied to soils globally (20). Here we used a knowledge of the atzA, -B, and -C gene sequences to investigate the presence of homologous genes in other atrazine-degrading bacteria. In this study, we report that five atrazine-degrading microorganisms, which were recently isolated from geographically separated sites exposed to atrazine, contained nearly identical atzA, -B, and -C genes. Open in a separate windowFIG. 1Pathway for atrazine catabolism to cyanuric acid in Pseudomonas sp. strain ADP.

Atrazine-catabolizing bacteria used in this study.

Until recently, attempts at isolating bacteria (18) or fungi (27) that completely degrade atrazine to carbon dioxide, ammonia, and chloride were unsuccessful. While several microorganisms were shown to dealkylate atrazine, they were unable to displace the chlorine atom (41, 54). Since 1994, several research groups have independently isolated atrazine-degrading bacteria that displaced the chlorine atom and mineralized atrazine (3, 7, 13, 35, 39, 46). Six of these bacterial cultures, listed in Table ​Table1,1, were studied here, and the Clavibacter strain had been investigated previously (13).

TABLE 1

Recently isolated atrazine-catabolizing bacteria

Phosphorylation of ATPase subunits of the 26S proteasome

The 26S proteasome complex plays a major role in the non-lysosomal degradation of intracellular proteins. Purified 26S proteasomes give a pattern of more than 40 spots on 2D-PAGE gels. The positions of subunits have been identified by mass spectrometry of tryptic peptides and by immunoblotting with subunit-specific antipeptide antibodies. Two-dimensional polyacrylamide gel electrophoresis of proteasomes immunoprecipitated from [³²P]phosphate-labelled human embryo lung L-132 cells revealed the presence of at least three major phosphorylated polypeptides among the regulatory subunits as well as the C8 and C9 components of the core 20S proteasome. Comparison with the positions of the regulatory polypeptides revealed a minor phosphorylated form to be S7 (MSS1). Antibodies against S4, S6 (TBP7) and S12 (MOV34) all cross-reacted at the position of major phosphorylated polypeptides suggesting that several of the ATPase subunits may be phosphorylated. The phosphorylation of S4 was confirmed by double immunoprecipitation experiments in which 26S proteasomes were immunoprecipitated as above and dissociated and then S4 was immunoprecipitated with subunit-specific antibodies. Antibodies against the non-ATPase subunit S10, which has been suggested by others to be phosphorylated, did not coincide with the position of a phosphorylated polypeptide. Some differences were observed in the 2D-PAGE pattern of proteasomes immunoprecipitated from cultured cells compared to purified rat liver 26S proteasomes suggesting possible differences in subunit compositions of 26S proteasomes.     

The Saccharomyces cerevisiae YCC5 (YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine

The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.     

ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tζζ– GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.