Paul M. Lizardi

Rapid isolation of RNA using proteinase K and sodium perchlorate

Abstract A simple, efficient procedure for the isolation of cellular nucleic acids is described. It combines the use of sodium dodecyl sulfate, proteinase K, sodium perchlorate, and isopropanol precipitation. The yields and purity of RNA extracted from a variety of sources are comparable or superior to those obtained by phenol extraction. High molecular weight RNA (ribosomal as well as nonribosomal) is recovered intact and in high yield. Fibroin messenger RNA ( M r 5.8 × 10 6 ) isolated by this procedure is biologically active.

The size of pulse-labeled fibroin messenger RNA

A method has been developed for the isolation of fibroin gene transcripts from total RNA of the silkworm Bombyx mori. It is based on affinity chromatography using Sephadex-bound polynucleotides capable of selectively hybridizing with fibroin mRNA sequences. In vivo pulse labeling of the posterior silk gland for periods of 10-35 min produces labeled heterogeneous nuclear RNA of high molecular weight (greater 40S). Fibroin gene transcripts can be selected from the total hnRNA population by two consecutive passages through the affinity column. Analysis of the column-bound material in denaturing polyacrylamide-agarose gels reveals that the size of pulse-labeled fibroin mRNA is essentially the same (within 5%) as that of mature cytoplasmic mRNA. This holds true for pulses as short as 6 min, where even nascent mRNA can be observed. However, a small shoulder of material is present on the heavy side of the pulse-labeled mRNA, which could be indicative of an extremely short-lived precurosr species. The purified pulse-labeled mRNA (10 min incorporation) has been further analyzed by chromatography in oligo(dT)-cellulose. The data show that the mRNA is polyadenylated within a few minutes after synthesis.

Preparative isolation of DNA and biologically active mRNA from diethylaminoethyl membrane

Abstract A simple method is described for the preparative isolation of both DNA and mRNA after electrophoretic separation in agarose gels. The method is based on the quantitative binding of the nucleic acid to a membrane containing charged diethylaminoethyl (DEAE) groups, followed by elution under high salt conditions. Fragments of bacteriophage λ cut with Hind III, ranging in size from 565 to 9536 bp were recovered at efficiencies ranging from 84% to 35%, respectively. Poly A + mRNAs from hen oviduct were recovered with an efficiency of 65–80%. Recovery of biological activity was about 70%.

Genetic polymorphism of silk fibroin studied by two-dimensional translation pause fingerprints

Abstract Silk fibroins from a number of B mori strains have been compared, and at least six different polypeptide size phenotypes, which show apparent molecular weights of 365,000–415,000 in SDS polyacrylamide gels, have been identified. Analysis of the corresponding fibroin messenger RNAs in denaturing gels shows that mRNA size is largely correlated with polypeptide length. The mRNAs vary in size from 5.50 × 10 6 to 6.30 × 10 6 daltons. It has been shown elsewhere that the translation of silk fibroin mRNA in a reticulocyte cell-free system proceeds discontinuously. In this paper, I demonstrate that this discontinuous translation phenomenon can be exploited to map the location of divergent amino acid sequences in fibroin variants. SDS gel analysis of translational pause patterns shows that divergence arises internally after a relatively long amino-terminal sequence which appears to be conserved. Two-dimensional gel analysis using V8 protease digestion in the second dimension produces fingerprints of fibroin peptide fragments ordered from the amino to the carboxyl terminus of the protein. These fingerprints provide additional evidence for extensive internal divergence of the fibroins and a reduced degree of divergence near the termini. A plausible explanation for the observed genetic variability is the occurrence of relatively large unequal crossing-over exchanges in the repetitive domain of the fibroin gene.

Biogenesis of Silk Fibroin mRNA: An Example of Very Rapid Processing?

Publisher Summary This chapter reviews that an affinity column containing a bound synthetic polynucleotide has been used as a tool for the isolation of pulse-labeled fibroin mRNA. Two passages through the column are sufficient to purify the pulse-labeled fibroin gene transcripts from the total hnRNA population. The fibroin mRNA molecules isolated after pulse-labeling in vivo are not demonstrably larger than fibroin mRNA. In fact, for short pulse-labeling times the peak of the gel profile of pulse-labeled mRNA is at a position one to two gel slices smaller than mature mRNA. To investigate the possibility that putative RNA processing cuts may be occurring even before the termination of transcription, experiments have been done under conditions in which processing may be expected to be slower. In vivo pulse-labeling of animals at 11.5°C, followed by affinity chromatography and gel electrophoresis, results in a different size-profile of newly synthesized fibroin mRNA. In this case, the peak of the size distribution is a few gel slices larger than the mature mRNA peak. This larger material, which accumulates at low temperature, could represent a very short-lived precursor species, and may in fact be the “primary” transcript of the silk fibroin gene.