Biomedical subjects

M Speek

Publications and source records attributed to M Speek.

3 recordsLinked to original sources

PubMed1992

Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting.

The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.

Amino Acid Sequence↗

PubMed1988

Smart2, a cosmid vector with a phage lambda origin for both systematic chromosome walking and P-element-mediated gene transfer in Drosophila.

We describe a new phage-lambda-replicon-based cosmid vector suitable for both chromosome walking and P-element-mediated transformation in Drosophila. Its unique BamHI cloning site is flanked by the promoters for the SP6 and T7-encoded RNA polymerases, permitting the synthesis of probes complementary to the ends of the cloned inserts for library screening. The selectable marker is tet for bacterial cell transformation and neo for Drosophila transformation expressed under the control of the Drosophila hsp70 promoter.

Animals↗

PubMed1986

Consecutive DNA terminator sequencing by using enzymatically generated primers.

An improved strategy for the dideoxy chain termination sequencing of M13 recombinant DNA using enzymatically generated primers is presented. It involves synchronous extension of the universal primer with the Klenow fragment of DNA polymerase I at an average rate of 200 deoxynucleoside triphosphates per minute to the immediate downstream region of a preselected restriction enzyme site. Subsequent cleavage with the restriction enzyme generates the short primers with homogeneous 5' ends which can be used for further sequencing. The next restriction sites are selected in the newly sequenced regions of DNA by means of a microcomputer. By repeating this primer extension-cleavage-sequencing strategy sequences altogether about 6 kb long from several recombinant single-stranded M13 DNAs have been determined by using twenty restriction enzymes with different specificities.

Base Sequence↗