A simplified solid-phase assay for the quantitation of native membrane proteins. Application to the measurement of EGF receptor induction by dexamethasone.

Direct application of membrane fractions to a nitrocellulose support without previous solubilization, denaturation or fixation of the antigen permits the quantitation of antibodies binding to denaturation sensitive epitopes. In experiments reported here, this method was used to determine the rate of EGF receptor expression on squamous carcinoma cell lines. The assay revealed that dexamethasone treatment leads to an eight-fold increase of EGF receptor protein expression on C4-I cervical carcinoma cells. The data, when compared to the results obtained in immunoprecipitation experiments, suggest that this simple protocol yields reliable and precise quantitative data. The simplicity of the method permits simultaneous testing of large sample numbers with various antibodies.

Efficient low redundancy large-scale DNA sequencing at EMBL.

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.

Detection of C trachomatis in urogenital specimens by polymerase chain reaction.

OBJECTIVE: To establish a polymerase chain reaction (PCR) protocol for the detection of urogenital C trachomatis infection and to compare it with the detection in cell culture. SPECIMENS: Urethral specimens were collected from 62 male patients and cervical specimens from 106 female patients. SETTING: Department of Dermatology and Venereology, Ruprecht-Karls-Universität, Heidelberg. METHODS: Urogenital specimens were simply boiled for 15 minutes and subsequently subjected to amplification without prior extraction of nucleic acid. The DNA sequence selected for amplification is located in the third open reading frame of the ubiquitous C trachomatis plasmid pCTT1. The amplified products were demonstrated by agarose gel electrophoresis followed by Southern blot hybridization. In addition, specimens were investigated with cell culture. MAIN OUTCOME MEASURES: Results of PCR and cell culture. RESULTS: PCR detected all C trachomatis serovars relevant for urogenital infections (D-L2). Serial dilution experiments revealed that the PCR procedure was 100 fold more sensitive than cell culture. The investigation of 168 urogenital specimens showed that the PCR confirmed all 30 cell culture positive results, however, out of the 138 cell culture negative specimens 16 were positive using the PCR. CONCLUSIONS: A substantial number of urogenital C trachomatis infections detectable by PCR may be missed by the cell culture technique.

Glucocorticoid hormones reduce the expression of major histocompatibility class I antigens on human epithelial cells.

Expression of a critical level of major histocompatibility complex (MHC) class I antigens on epithelial cells is a prerequisite for the action of specific cytolytic immune response cells. Glucocorticoid hormones have strong immunosuppressive effects. Therefore, we investigated the influence of the synthetic glucocorticoid dexamethasone on the expression level of MHC class I antigens on human epithelial cell lines. Long-term treatment with dexamethasone leads to reduced MHC class I surface antigen expression and to decreased total membrane-bound MHC class I protein. The steady-state mRNA level is significantly decreased and the transcription rate of MHC class I genes is reduced.

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.