M Aldea

Transcript mapping using [35S]DNA probes, trichloroacetate solvent and dideoxy sequencing ladders: a rapid method for identification of transcriptional start points

A simple method for RNA transcript mapping has been developed that combines the use of 35S-labeled M13 DNA probes and the presence of high concentrations of sodium trichloroacetate in the hybridization buffer. These hybridization conditions permit the use of M13 probes without purification from the template. The dideoxy sequencing ladders used for sizing the protected DNA fragments are obtained from the same M13 templates utilized to synthesize the DNA probes. The method was tested by analyzing the transcripts controlled by lac, ptr and trxA promoters. Comparison of the results with previously published data obtained with the conventional S1 nuclease mapping technique indicated that the present method is just as precise and at least 50 times more sensitive. Clones constructed for sequencing a gene of interest can be used directly to identify transcriptional start points.

Generation of a detailed physical and genetic map of the ilv-metE-udp region of the Escherichia coli chromosome☆

The entire ilv-metE-udp region of the Escherichia coli chromosome has been cloned in two steps using the lambda replacement vector EMBL4. A detailed restriction map for approximately 70 X 10(3) bases of DNA has been generated. The gpp and udp structural genes have been identified, the cya and metE genes have been physically located, and the direction of recQ gene transcription has been determined. By examining a variety of plasmid subclones, 44 polypeptides have been detected using maxicell and minicell analysis, accounting for 70% of the maximum coding capacity of the entire region. On the basis of the observed gene density in the ilv-metE-udp region, a total number of 3000 genes is predicted for the entire E. coli chromosome. In addition, anomalies in cotransduction frequencies that have been observed in this region have been interpreted by employing a new formula that incorporates the effects of different transducing fragment representations and recombination probabilities.

CLONING: a microcomputer program for cloning simulations

A comprehensive computational tool is presented that performs cloning simulations using IBM PC/XT/AT or compatible microcomputers. The CLONING program contains a specific data base for restriction sites, gene markers, fragment sources and reference comments. It draws complete linear or circular maps either on the screen or employing conventional dot-matrix printers. The design of new recombinant molecules is a totally interactive process.

Instructions for the CLONING program

These instructions for CLONING were developed to assist the user in understanding the operation of the program [Aldea and Kushner, Gene 65 (1988) 111-116]. The program provides a computational tool that performs cloning simulations using IBM PC/XT/AT or compatible microcomputers. The design of new recombinant molecules is a totally interactive process.