Search results for " DNA"

Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors

2014

Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infe…

Plasmid conjugation from Proteobacteria as evidence for the origin of xenologous genes in Cyanobacteria

2014

Comparative genomics have shown that 5% of Synechococcus elongatus PCC 7942 genes are of probable proteobacterial origin. To investigate the role of interphylum conjugation in cyanobacterial gene acquisition, we tested the ability of a set of prototype proteobacterial conjugative plasmids (RP4, pKM101, R388, R64, and F) to transfer DNA from Escherichia coli to S. elongatus. A series of BioBrick-compatible, mobilizable shuttle vectors was developed. These vectors were based on the putative origin of replication of the Synechococcus resident plasmid pANL. Not only broad-host-range plasmids, such as RP4 and R388, but also narrower-host-range plasmids, such as pKM101, all encoding MPFT-type IV …

Correlation of plasmid DNA supercoiling and the efficiency of plasmid gene transcription

2007

Immunological Methods for Analysis of Recombinant Proteins

1998

We want to introduce researchers to techniques that help to solve some problems in the work of the molecular biologist. After transformation of recombinant DNA in E. coli cells many clones are usually produced, and the same situation appears if recombinant DNA expression libraries are available. Furthermore, if appropriate monoclonal or polyclonal antibodies are available, the method of immunoscreening of colonies for direct immunological detection of translational products of cloned genes can be used. Selected clones could be chosen for further studies such as determination of their primary structure by DNA sequencing and for characterization of an appropriate expressed protein.

Use of the Transglutaminase Reaction To Study the Dissociation of Histone N-Terminal Tails from DNA in Nucleosome Core Particles

1997

We have recently shown that core histones are glutaminyl substrates for transglutaminase (TGase) and that when native nucleosome cores are incubated with monodansylcadaverine (DNC) as donor amine, this fluorescent probe is incorporated into Gln5 and Gln19 of H3 and in Gln22 of H2B [Ballestar et al. (1996) J. Biol. Chem. 271, 18817-18825]. In the present paper, we report that the cause by which Gln22 of H2B is modified in nucleosomes but not in the free histone is the interaction of the region containing that glutamine with DNA. We have used the specificity of the TGase reaction to study the changes induced by increasing ionic strength in the interaction between the histone N-terminal tails …

Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients

2011

Hoerning A, Kalkavan H, Rehme C, Menke J, Worm K, Garritsen HSP, Buscher R, Hoyer PF. Quantitative real-time ARMS-qPCR for mitochondrial DNA enables accurate detection of microchimerism in renal transplant recipients. Pediatr Transplantation 2011: 15: 809–818. © 2011 John Wiley & Sons A/S. Abstract:  The presence of microchimerism in peripheral blood of solid organ transplant recipients has been postulated to be beneficial for allograft acceptance. Kinetics of donor cell trafficking and accumulation in pediatric allograft recipients are largely unknown. In this study, we implemented SNPs of the HVRs I and II of mitochondrial DNA to serve as molecular genetic markers to detect donor-specific…

313: A cDNA-based assay for donor-chimerism analysis of epidermal langerhans cells

2007

Structural and evolutionary analysis of the copia-like elements in the Arabidopsis thaliana genome.

2001

The analysis of 460 kb of genomic sequence of Arabidopsis thaliana chromosome III allowed us to identify two new transposable elements named AtC1 and AtC2. AtC1 shows identical long terminal repeats (LTRs) and all the structural features characteristic of the copia-like active elements. AtC2 is also a full copia-like element, but a putative stop codon in the open reading frame (ORF) would produce a truncated protein. In order to identify the copia-like fraction of the A. thaliana genome, a careful computer-based analysis of the available sequences (which correspond to 92% of the genome) was performed. Approximately 300 nonredundant copia-like sequences homologous to AtC1 and AtC2 were detec…

The Norway spruce genome sequence and conifer genome evolution

2013

Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinu…

Common genomic structure for the Lepidoptera cadherin-like genes.

2005

A cadherin-like protein present in the midgut epithelial cells of Lepidoptera is associated with insect resistance to Bacillus thuringiensis Cry toxins. We describe for the first time the genes that encode the cadherin-like proteins in Ostrinia nubilalis, Helicoverpa armigera, and Bombyx mori, and analyze their organization. These genes encompass 19.6 kb, 20.0 kb, and 41.8 kb of genomic DNA, respectively, and despite the size heterogeneity, they are all composed of 35 exons that are linked by 34 introns. In contrast to the high variability noted for the sizes of the introns, the sizes of the coding exons were almost completely preserved among the three species, because the intronic sequence…