Stephanie Wittig

Heat shock gene expression is regulated during teratocarcinoma cell differentiation and early embryonic development.

Abstract Undifferentiated teratocarcinoma stem cells do not express heat shock genes. Solid teratocarcinomas grown in vivo which contain clusters of teratocarcinoma-derived differentiated tissue do respond to heat shock. During mouse embryonic development the expression of heat shock genes is first observed with morula/blastocyst stages of mouse primplantation embryos.

Function of a tRNA gene promoter depends on nucleosome position.

We have used an in vitro procedure to direct nucleosomes to different sites on a chicken embryo tRNA gene. The results show that transcription of the tRNA gene depends on nucleosome position and in a functional promoter structure, the constant gene sequence TTCGA coincides with the nucleosome middle axis.

Stabilization of R loop structures by photochemical crosslinking with 4,5′,8-trimethylpsoralen: Application to gene enrichment and molecular cloning

Abstract R loops formed between nucleosomal DNA and tRNA can be photochemically crosslinked with 4,5′,8-trimethylpsoralen directly in the R loop formation buffer. When biotin is coupled to the tRNA 3′-terminus via a diaminohexan linker and the modified tRNA employed for R loop hybridization the crosslinked R loops can be efficiently purified by affinity chromatography on avidin-glass columns. Following tRNA hydrolysis the partially crosslinked double stranded DNA, highly enriched for tRNA genes can be cloned in E. coli χ1776.

The enrichment of chicken embryo tRNA genes in nucleosomal DNA by reversed phase chromatography

The RPCJ reversed phase chromatography system is well known in the high resolution fractionation of tRNA into individual tRNA species [l-3]. The system combines phase distribution effects on a hydrophobic polychlorotrifluoroethylene matrix with ion exchange chromatography accomplished by the tricaprylylmethylammonium groups coated onto the matrix. It follows that besides common ionic binding forces the extent of secondary structure affecting the accessibility of purineand pyrimidine-base charges modulates the influence of each of the two chromatographic effects on a particular class of nucleic acids. In the RPCJ chromatography of restriction fragments of double stranded DNA a number of fragments elute at positions not expected in view of their molecular weight, i.e., their ionic net charge, signalling the influence of the phase distribution effect [4,5]. RNAs with little or no secondary structure elute preferentially in order of their size under the main influence of ion exchange forces from RPC-5 columns [6]. Here we report on two methods for the enrichment of tRNA genes in nucleosomal DNA employing extensions of both chromatographic effects of RPC-5: 1. DNA of 570 basepairs isolated from nucleosome trimers [7] was fractionated by RPC-5 using concentration gradients of the G-C-specific dye phenyl neutral red [8 3 and of sodium acetate, simultaneously. Assaying the effluent DNA fractions for hybridization with ‘2sI-labelled chicken embryo total tRNA revealed distinct