Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.

Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.

A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.

https://www.pnas.org/content/pnas/84/21/7413.full.pdf

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure

A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N′-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full-sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10656.x

An efficient mRNA-dependent translation system from reticulocyte lysates.

A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.

Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells.

Extracts prepared from commercially available wheat germ efficiently translate messenger RNAs from either viral or eukaryotic origin. The addition of tobacco mosaic virus RNA stimulated amino-acid incorporation more than 100-times and rabbit globin 9S RNA between 20- and 30-times. The in vitro product directed by globin 9S RNA comigrated precisely with authentic rabbit globin on sodium dodecyl sulfate-polyacrylamide gels. Furthermore, during high voltage ionophoresis two [35S]methionine-labeled tryptic peptides synthesized in vitro comigrated in two dimensions with αT5 and βT5 tryptic peptides from authentic [35S]methionine-labeled rabbit globin.

https://www.pnas.org/content/pnas/70/8/2330.full.pdf

Efficient Translation of Tobacco Mosaic Virus RNA and Rabbit Globin 9S RNA in a Cell-Free System from Commercial Wheat Germ

https://www.cell.com/cell/pdf/0092-8674(84)90440-9.pdf

Construction and applications of a highly transmissible murine retrovirus shuttle vector.

We present a simple method for directly correlating structural gene sequences in DNA with their corresponding mRNAs. This is based upon the fact that mRNA hybridized with its complementary DNA will not direct the cell-free synthesis of a complete polypeptide. Full translational activity of the mRNA is recovered upon the heat melting of the hybrid. Utilizing the rabbit beta globin clone PbetaG1, we demonstrate the application of hybrid-arrested translation for the identification of structural gene sequences within recombinant DNA molecules. In addition, the method is used to locate and order precisely several adenovirus 2 polypeptides within the viral genome.

https://www.pnas.org/content/pnas/74/10/4370.full.pdf

Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation

We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation

Simian adenovirus 7 (SA7) is a highly oncogenic virus, capable of causing tumors in hamsters upon the direct injection of viral DNA. We determined the transcriptional organization of the transforming region and compared it with that of the human adenoviruses. This analysis demonstrated that there are two independently promoted transcription units similar to the E1a and E1b regions of the human adenoviruses. The nucleotide sequence of the SA7 E1a region demonstrated considerable homology with the human adenoviruses, both in the sequences that regulate E1a expression and in the encoded polypeptides. The amino acid homology was reflected in the ability of SA7 to complement the growth of human adenoviruses mutant in the E1a region. Furthermore, we found two regions of amino acid homology unique to SA7 and the highly oncogenic human adenovirus 12.

Bryan E. Roberts

Papers

Efficient Translation of Tobacco Mosaic Virus RNA and Rabbit Globin 9S RNA in a Cell-Free System from Commercial Wheat Germ

Construction and applications of a highly transmissible murine retrovirus shuttle vector.

Structural gene identification and mapping by DNA-mRNA hybrid-arrested cell-free translation

Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation

E1a regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related.