Synthesis of cytochrome c oxidase polypeptides in an Escherichia coli cell-free system directed by Saccharomyces cerevisiae mitochondrial DNA.

Summary Using purified yeast mitochondrial DNA as a template forE. coli RNA polymerase (holoenzyme) complementary mitochondrial RNA has been synthesized in vitro. This RNA has been used to direct a low backgroundE. coli S-30 protein-synthesizing system. The synthesis of mitochondrial polypeptides has been detected by using antiserum raised against purified cytochromec oxidase holoenzyme and shown to be specific for this antigen. The antiserum-antigen complex was dissociated and subject to SDS-polyacrylamide gel electrophoresis and the presence of 3 polypeptides of 39, 31, and 26×10³ daltons molecular weight demonstrated, which correspond to the subunits synthesized by mitochondria in whole cells which are inhibited with cycloheximide.     

Proteins synthesized by a non-induced bacteriocinogenic factor in minicells of Escherichia coli

Summary The cloacinogenic factor Clo DF13 from Enterobacter cloacae has been transferred to the minicell-producing strain P678-54 of Escherichia coli K12. The data presented show that this Clo DF13 factor segregates into minicells of P678-54 (Clo DF13) and that this factor is the only plasmid, present in these minicells. Proteins from purified P678-54 (Clo DF13) and P678-54 minicells, previously labelled with ¹⁴C-amino acids, were compared after electrophoresis on SDS-polyacrylamide gels. From this comparison it appeared that a noninduced Clo DF13 factor directs the synthesis of 4 proteins. The molecular weights of these proteins could be estimated to be about 72000, 32000, 18500 and 12000. In P678-54 (Clo DF13) minicells, one additional Clo DF13 protein was found to be unlabelled. Apparently this protein is not synthesized in P678-54 (Clo DF13) minicells, but is segregated into or is attached to the minicells after being synthesized in the P678-54 (Clo DF13) cells. The molecular weight of this protein is about 62000, which corresponds to the molecular weight of cloacin.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae Cell Lysates

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp^r gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per μg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.     

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp(r) gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.     

Metalloenzymes in DNA repair. Escherichia coli endonuclease IV and Saccharomyces cerevisiae Apn1.

Escherichia coli endonuclease IV and its Saccharomyces cerevisiae homologue Apn1, two DNA repair enzymes for free radical damages, were previously shown to be inactivated by metal-chelating agents. In the present study, atomic absorption spectrometry of endonuclease IV revealed the presence of 2.4 zinc and 0.7 manganese atoms, whereas Apn1 contained 3.3 zinc atoms and no significant manganese. EDTA-inactivated endonuclease IV retained 0.7 zinc atom but little detectable manganese. ZnCl2 reactivated 1,10-phenanthroline-treated Apn1, but was ineffective with endonuclease IV treated with either 1,10-phenanthroline or EDTA. In contrast, enzymatic activity was restored to both enzymes after EDTA treatment by incubation with CoCl2 and to a lesser extent by MnCl2. Endonuclease IV, reactivated with CoCl2 or MnCl2, regained all of the activities characteristic of the native enzyme. MnCl2 was as effective as CoCl2 at restoring activity to the 1,10-phenanthroline-treated enzymes. The results indicate that intrinsic metals play critical roles in both endonuclease IV and Apn1 and that manganese may perform a special function in endonuclease IV. Possible mechanistic roles for the metals in these DNA repair enzymes are discussed.     

Mapping of Escherichia coli RNA polymerase binding sites on 2-micrometers DNA from Saccharomyces cerevisiae. Heterogeneity within the inverted duplication and evidence for an eukaryotic invertible DNA sequence.

The 2-μm DNA plasmids from Saccharomyces cerevisiae strain H1 and strain HQ/5C were analyzed by electron microscopy for the presence of Escherichia coli RNA polymerase binding sites. On native 2-μm DNA isolated from strain HQ/5C five RNA polymerase binding sites were detected. One further site was mapped on cloned 2-μm DNA type 23 from S. cerevisiae strain H1. This additional site is located at a distance of 2.15 kilobases from EcoRI site B inside one of the inverted duplication (id) sequences. No such binding site could be detected in the other id sequence of the type 23 molecule, thus indicating that the two id sequences of strain H1 differ in at least one short region. The location of the id sequence carrying the RNA polymerase binding site was analyzed in native 2-μm DNA isolated from strain H1 and found to be present on HindIII fragment 2 and absent from HindIII fragment 5. This indicates that at least a part of the id sequences has a fixed position with respect to the unique S segment and further suggests a site specific recombination mechanism for the inversion of one of the unique segments. As a control for the specificity of RNA polymerase binding, we have mapped binding sites on vectors pBR313 and pBR322. The location of the E. coli RNA polymerase binding sites on 2-μm DNA is discussed in relation to the DNA regions expressed in E. coli minicells.     

Expression of recombinant DNA functional products in Escherichia coli anucleate minicells

This review covers the use of anucleate minicells of Escherichia coli for expressing the recombinant DNA encoded proteins. We briefly discuss the methods being used for preparation of anucleate minicells, incorporation of cloned DNA and assessment of gene expression. While the largest use has been that of microbially derived cloned functional DNA, examples of eukaryotic gene product synthesis have also been reviewed. This technology may represent some interesting commercial opportunities.     

Examination of newly synthesized DNA in Escherichia coli.

Summary The short (0-20S) Okazaki fragments seen upon pulse-labeling E. coli (thy ⁺, deo ⁺) with ³H-thymidine are actually composed of three types of DNA fragments: (1) true replication intermediates, (2) post-replication repair fragments (such as those which arise subsequent to the removal of misincorporated uracil), and (3) chromosomal fragments. Our experiments show that the number of pulse-labeled fragments decreases slightly with the introduction of the ung-1 mutation into E. coli K-12 (dut ⁺, thyl ⁺, polA ⁺), and that there are fewer fragments found in E. coli B/r than in E. coli K-12 ung-1. This suggests that while some fraction of pulse-labeled fragments may be due to repair, this fraction can vary among different strains; moreover, the majority of fragments appear to be replication intermediates. Selfhybridization (reannealing) of pulse-labeled fragments from E. coli B/r show that these fragments are asymmetric with respect to the strand origin and with respect to their size: the smaller (3-8S) fragments come from only one of the parental strands, whereas the larger (13-20S) fragments are synthesized equally from both parental strands. We interpret our results to mean that replication can be discontinuous on both strands but asymmetric with respect to both the size of the fragments and the size of the discontinuous region on the two strands.     

Nucleoside triphosphate pools in minicells of Escherichia coli.

The nucleoside triphosphate pools of Escherichia coli minicells are different from those in parental cells. The growth phase in which minicells accumulate significantly affects the pool sizes.     

Rapid isolation of Escherichia coli minicells by glass-fiber filtration: study of plasmid-coded polypeptides

A filtration technique is described to purify Escherichia coli chi 1488 minicells much more rapidly than the usual method involving sucrose gradient centrifugation, and to produce minicells that have not been subjected to osmotic stress. The minicells so prepared are metabolically active as indicated by the in vivo incorporation of [35S]methionine into plasmid-coded polypeptides.     

Mucopeptide biosynthesis by minicells of Escherichia coli.

Minicells produced by Escherichia coli chi925 incorporated amino acids and N-acetyl-d-glucosamine into mucopeptide.     

Method for localization of cloned DNA fragments on the Escherichia coli chromosome.

In exponentially growing cultures of Escherichia coli strains carrying the dnaC28 mutation, DNA replication can be synchronized by temperature changes (R. L. Rodriguez, M. S. Dalbey, and C. I. Davern, J. Mol. Biol. 74:599-604, 1973). We used this synchronization procedure and DNA-DNA hybridization to develop a technique for the localization of cloned chromosomal fragments on the genetic map. Because of the bidirectional nature of replication in E. coli, our method gave two possible positions (one on each replication arm). However because of the precision obtained for each position (+/- 1 map unit), the final mapping with various genetic techniques was greatly facilitated. Using this technique and a simple chromosomal mobilization test, we located at 93.2 +/- 1 min a cloned DNA fragment carrying an extragenic suppressor of dnaA46, a thermosensitive mutation in the dnaA initiation gene. Further analysis showed that the groES (mopA) and groEL (mopB) genes, both located at 94.2 min on the standard map, were indeed carried by the cloned suppressor fragment.     

Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae.

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.     

Expression of a Saccharomyces cerevisiae photolyase gene in Escherichia coli.

A 3.3-kilobase PvuII fragment carrying the PHR1 gene of Saccharomyces cerevisiae has been cloned into an Escherichia coli expression vector and introduced into E. coli strains deficient in DNA photolyase. Complementation of the E. coli phr-1 mutation was observed, strongly suggesting that the yeast PHR1 gene encodes a DNA photolyase.