Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA.

Reverse transcriptase (RT) is the key enzyme required for conversion of RNA to DNA. Cloning of Moloney murine leukemia virus (MMLV) RT has enable engineering an RT that lacks endogenous RNase H activity. RT catalyzes cDNA synthesis more efficiently in the absence of RNase H. We describe here a number of properties of MMLV RT and RNase H-minus MMLV RT not summarized in a single location elsewhere, providing a basis for best use of these enzymes in cDNA synthesis. In addition, general guidelines and detailed protocols are provided for use of MMLV RTs in one tube double-stranded cDNA synthesis, in [32P]cDNA synthesis, and in RT-PCR and long RT-PCR.

The isolation of differentially expressed genes in fibroblast growth factor stimulated BC3H1 cells by subtractive hybridization.

We have developed a subtractive hybridization procedure based on the hybridization of a single-stranded phagemid cDNA library (target) to biotinylated RNA (driver). We have applied this method to fibroblast growth factor (FGF) induced-uninduced mouse brain tumor-derived muscle-like cell. BC3Hl cDNA libraries. After hybridization to a C(o)t value of 1000, cDNAs common to the target and driver populations were subtracted up to 231-fold, whereas several highly induced genes were enriched from 2-15-fold. Interestingly, moderately induced genes (e.g., the 12-fold-induced nur/77 gene) were subtracted even at a low C(o)t value of 50. Therefore, at every C(o)t tested, subtractive hybridization tended to equalize the uninduced and moderately induced common sequences within target populations regardless of the abundance of the gene species. These observations suggest that subtractive hybridization should only be used for identifying target genes that are either uniquely expressed or highly induced.

Enrichment for 5'-TG termini: a method for subcloning structural genes into expression vectors.

We describe a method for creating a population of randomly digested, blunt-ended DNA fragments with 5'-TG... 3'-AC... (TG) at their termini. When these fragments were ligated to a blunt-ended vector that contains ...CATA-3' ...GTAT-5' at its termini, as high as 84% of the clones obtained after transformation contained plasmids with a reconstructed Nde I site ... CATATG... ...GTATAC.... When the DNA vector is prepared from an appropriate plasmid [Gross et al., Mol. Cell. Biol. 5 (1985) 1015-1024; Kotewicz et al., Gene 35 (1985) 249-258], the ATG within the restriction site corresponds to a start codon positioned downstream from a strong ribosome-binding site and controllable promoter. If a gene has been digested to the TG contained within its authentic initiation codon, the endogenous translation-initiation control sequences are deleted and expression can be controlled using the plasmid-derived promoter. In addition, a gene digested to other in-frame TGs can potentially express proteins with altered N termini. Using this method, we have placed the structural gene of SP6 RNA polymerase, trimmed precisely to its authentic start codon, under the control of the tac promoter.

Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase.

A simple method for generating cDNA libraries has been described (1) in which RNase H-DNA polymerase I-mediated second-strand cDNA synthesis primes from an RNA oligonucleotide derived from the 5' (capped) end of mRNA. The size of this oligonucleotide and the fate of the information corresponding to the RNA during subsequent cloning have not been established. We show here that the 5'-most RNA primer varies in length from 8 to 21 nucleotides, and that information corresponding to the length of the RNA primer is normally lost during cloning. A modification of the second-strand cDNA synthesis procedure is described which allows cloning of all, or almost all, of the primer sequence information. In addition, we show that the presence of E. coli DNA ligase during second-strand cDNA synthesis can increase the length of the cDNA clones obtained from long RNAs. Cloning by addition of linkers provides the greatest chance of obtaining near full-length cDNA clones from long mRNAs.

Isolation of cloned Moloney murine leukemia virus reverse transcriptase lacking ribonuclease H activity.

Retroviral reverse transcriptase possesses DNA polymerase and ribonuclease H (RNase H) activity within a single polypeptide. Chemical or proteolytic treatment of reverse transcriptase has been used in the past to produce enzyme that is missing DNA polymerase activity and retains RNase H activity. It has not been possible to obtain reverse transcriptase that lacks RNase H but retains DNA polymerase activity. We have constructed a novel deletion derivative of the cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase gene, expressed the gene in E. coli, and purified the protein to near homogeneity. The purified enzyme has a fully active DNA polymerase, but has no detectable RNase H activity. These results are consistent with, but do not prove, the conclusion that the DNA polymerase and RNase H activities of M-MLV reverse transcriptase reside within separate structural domains.

Influence on stability in Escherichia coli of the carboxy-terminal structure of cloned Moloney murine leukemia virus reverse transcriptase.

We have cloned and expressed in Escherichia coli a section of the Moloney murine leukemia virus (Mo-MLV) pol gene which includes the entire coding region of mature reverse transcriptase (RT) plus 284 additional base pairs 3' to the coding region (Kotewicz et al., 1985). To prepare cloned Mo-MLV RT as close as possible to authentic RT in structure and activity, the universal terminator sequence GC(TTAA)3GC was introduced at a number of positions inside and outside the RT coding region within 200 nucleotides of its 3' end. The level of RT activity expressed from these constructs varied sevenfold. This variation was found to be directly related to the stability of the RT protein products in the E. coli K-12 strain K802; half-lives varied from 2 to 35 min. The stability of most of the RT proteins was not increased in E. coli K802 lon- cells, with the exception of two, whose half-lives were increased by a factor of two.

Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli.

A pBR322-derived expression vector, plasmid pKD1, was constructed containing the strong leftward promoter (pL) of bacteriophage lambda, the ribosome-binding site (RBS) of the cII gene of lambda, and a unique downstream NdeI restriction site for construction of an ATG initiation codon. The section of the pol gene of Moloney murine leukemia virus (M-MLV) that codes for reverse transcriptase (RT) was cloned into the NdeI site of this vector generating the plasmid pRT103. Upon thermal induction, enzymatically active RT was expressed in Escherichia coli [pRT103]. The identity of this activity was confirmed by its template specificity and its sensitivity to inhibition by immunoglobulin G (IgG) prepared against authentic murine RT. RT represented 20% of the newly synthesized protein in these cells 20 min after induction.

In vitro evidence that eukaryotic ribosomal RNA transcription is regulated by modification of RNA polymerase I.

We have utilized a cell-free transcription system from Acanthamoeba castellanii to test the functional activity of RNA polymerase I and transcription initiation factor I (TIF-I) during developmental down regulation of rRNA transcription. The results strongly suggest that rRNA transcription is regulated by modification, probably covalent, of RNA polymerase I: (1) The level of activity of TIF-I in extracts from transcriptionally active and inactive cells is constant. (2) The number of RNA polymerase I molecules in transcriptionally active and inactive cells is also constant. (3) In contrast, though the specific activity of polymerase I on damaged templates remains constant, both crude and purified polymerase I from inactive cells have lost the ability to participate in faithful initiation of rRNA transcription. (4) Polymerase I purified from transcriptionally active cells has the same subunit architecture as enzyme from inactive cells. However, the latter is heat denatured 5 times faster than the active polymerase.

Ribosomal ribonucleic acid repeat unit of Acanthamoeba castellanii: cloning and restriction endonuclease map.

The repeat unit coding for the precursor to 18S, 5.8S, and 26S ribosomal ribonucleic acids (rRNAs) has been cloned from the free-living soil amoeba Acanthamoeba castellanii. The cloned deoxyribonucleic acid (DNA) was mapped with 11 restriction endonucleases and by R-loop mapping. The entire repeat unit is 12 kbp (kilobase pairs) in length and contains sites for EcoRI, SmaI, BglII, SstI, Bam-HI, PstI, KpnI, HindIII, and XbaI but not for XhoI or SalI. All of the repeat units in the nuclear DNA appear to be identical, and no introns were detected. However, the regions which code for the two RNAs which comprise the 26S RNA are separated by a gap of approximately 200 base pairs. Unlike some other lower eukaryotes, the 5S RNA gene is not linked to this repeat unit. A fragment of the repeat unit which contains the initiation sequence of the putative precursor has been subcloned into pBR322 for use in vitro transcription studies.

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes.

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.

DNA-dependent RNA polymerases from Artemia salina. IV. appearance of nuclear RNA polymerase activity during pre-emergence development of encysted embryos.

Dessicated and encysted gastrulae of the brine shrimp Artemia salina remain metabolically dormant until they are rehydrated. At this time development resumes, culminating in the hatching of free swimming nauplius larvae. The resumption of embryogenesis provides a convenient system for studying biochemical events which accompany development of a eukaryotic organism, and in particular Artemia has proven useful for studies of the transcriptional regulation of gene expression. Encysted gastrulae of Artemia yielded only trace amounts of DNA-dependent RNA polymerase activity when crude nuclear pellets were subjected to sonication at high ionic strength. Furthermore, when crude nuclear pellets from encysted gastrulae and developing nauplius larvae were mixed prior to sonication, subsequent solubilization of proteins from the mixture did not yield RNA polymerase activity; sonication of the pellet from nauplii alone resulted in the solubilization of large quantities of RNA polymerases I and II as we have previously found [1]. RNA polymerases I and II were detectable in sonicates of crude nuclear pellets after 1-h incubation of Artemia cysts in sea water. This presents the possibility that dormant gastrulae of the brine shrimp contain RNA polymerase which is inactive, and that the rapid appearance of nuclear enzymatic activity which accompanies the resumption of development may not require de novo synthesis of the polymerase.