Mary Ann D. Brow

We present the complete nucleotide sequence of a cDNA encoding rat cyclophilin. The 743-nucleotide sequence contains a 42-nucleotide 5' noncoding region, a 492 nucleotide open reading frame corresponding to a translation product of 164 amino acids with a molecular weight of 17,874, and a 3' noncoding region of 209 nucleotides. Primer extension studies reveal the presence of one minor and two major transcription start sites. Southern blot analyses are consistent with as many as 20 copies of the cyclophilin gene and possible pseudogenes. Cyclophilin mRNA is expressed in virtually all types of tissues of rat and monkey and appears to have been highly conserved during mammalian evolution.

The highly thermostable DNA polymerase from Thermus aquaticus (Taq) is ideal for both manual and automated DNA sequencing because it is fast, highly processive, has little or no 3'-exonuclease activity, and is active over a broad range of temperatures. Sequencing protocols are presented that produce readable extension products greater than 1000 bases having uniform band intensities. A combination of high reaction temperatures and the base analog 7-deaza-2'-deoxyguanosine was used to sequence through G + C-rich DNA and to resolve gel compressions. We modified the polymerase chain reaction (PCR) conditions for direct DNA sequencing of asymmetric PCR products without intermediate purification by using Taq DNA polymerase. The coupling of template preparation by asymmetric PCR and direct sequencing should facilitate automation for large-scale sequencing projects.

Previously known 5' exonucleases of several eubacterial DNA polymerases have now been shown to be structure-specific endonucleases that cleave single-stranded DNA or RNA at the bifurcated end of a base-paired duplex. Cleavage was not coupled to synthesis, although primers accelerated the rate of cleavage considerably. The enzyme appeared to gain access to the cleavage site by moving from the free end of a 5' extension to the bifurcation of the duplex, where cleavage took place. Single-stranded 5' arms up to 200 nucleotides long were cleaved from such a duplex. Essentially any linear single-stranded nucleic acid can be targeted for specific cleavage by the 5' nuclease of DNA polymerase through hybridization with an oligonucleotide that converts the desired cleavage site into a substrate.