Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids.

A modified in vitro replication system has been characterized and used to catalogue the host proteins required for the replication of plasmid RSF1030. These extracts differ from systems described previously in that endogenous DNA is removed. Replication in vitro therefore requires an exogenouos RSF1030. Synthesis in the in vitro system faithfully mimics in vivo replication with respect to the products synthesized, effects of specific inhibitors, and requirements for RNA polymerase and DNA polymerase I. In addition, we find that proteins encoded by dnaB, dnaC, dnaG, dnaI, dnaP and polC (DNA polymerase III), are required for in vitro plasmid synthesis. The product of dnaA is not required. Extracts prepared from E. coli mutants deficient in in vitro replication can be complemented by addition of purified proteins or of extracts carrying the wild type protein.     

An improved method for detecting Y chromosomal DNA

Summary The DNA probe Y97 was derived from a repeat sequence in the human Y centromere, a region which must be present in a mitotically functional Y chromosome. We have demonstrated that Y97, which detects a Y-specific 5.5-kb Eco RI fragment by Southern analysis, is very useful for the molecular detection of small amounts of Y-derived material and represents a significant improvement over previous tests for molecular diagnosis of sex. The male-female difference in hybridization was unequivocal even when only 25 ng of total DNA was used per lane. Furthermore, in mixing experiments the 5.5-kb Eco RI fragment was detectable even when only 5% of the total DNA was male. By increasing hybridization stringency, we have developed a rapid, sensitive, and accurate method to detect Y chromosomal DNA in unrestricted samples.     

High-molecular-weight linear multimer formation by single-stranded DNA plasmids in Escherichia coli.

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.     

An improved bulk purification method for Escherichia coli elongation factor, Ts

A bulk purification procedure has been designed to maximize the yield of Escherichia coli elongation factor, Ts, with a minimum of effort and time. The enzyme purification is achieved by DEAE-Sepharose and elongation factor Tu-affinity chromatographies. The typical yield is 150 mg/kg of E. coli (B) cells.     

Loss of plasmids during enrichment for Escherichia coli.

Enrichment with sodium lauryl sulfate and incubation at 44.5 degrees C resulted in a loss of plasmids and decreased efficiency in the recovery of pathogenic. Escherichia coli strains from foods.     

Two-step method for curing Escherichia coli of ColE1-derived plasmids

To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn10, fadAB::Tn10 or other transposon insertions near polA. Reciprocal transduction to Met(+) Pol(+) or to Fad(+) Pol(+) ensures reestablishment of the original genotype except for loss of the plasmid. A set of useful bacterial strains is provided.     

An improved method for the purification of DNA-dependent RNA polymerase from Escherichia coli

DNA-dependent RNA polymerase from Escherichia coli was purified further by elution through heparin-Sepharose CL-6B column after the enzyme was obtained, partially purified, using Burgess and Jendrisak's method [(1975)Biochemistry 14, 4634] The total yield of the pure protein was 10 mg from 50 g of E.coli cells. The method was found to be very reproducible and convenient. The enzyme preparation had 60% active molecules and the elongation rate of RNA synthesis by this enzyme was measured to be 11 bases/s over delta D111 T7 DNA.     

Shuttle plasmids for Escherichia coli and Clostridium perfringens.

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.     

Plasmid pKKH: An improved vector with higher copy number for expression of foreign genes in Escherichia coli

Summary An improved vector of 2889 bp was constructed by mutation of copy number control system, into which foreign genes without or with the start codon ATG can be directly inserted for high-level expression in Escherichia coli. Deletion of the rop gene encoding a negative regulation protein ROP leads to increase the plasmid copy number, and finally makes the vector increase expression level more than 60% in comparison with initial one.     

An improved method for the purification of Bacillus subtilis glucose dehydrogenase cloned in Escherichia coli

An improved and simplified purification procedure has been developed for the isolation of the Bacillus subtilis glucose dehydrogenase which has resulted in a 10 fold higher yield of pure enzyme. The purification procedure utilizes gene cloning and an additional ammonium sulfate step to facilitate the removal of contaminating proteins. The procedure requires fewer chromatographic steps than previously reported, thus simplifying the procedure. This improved and simplified purification of B. subtilis glucose dehydrogenase will facilitate further structure-function studies of this sporulation specific enzyme.     

A new method for protein coexpression in Escherichia coli using two incompatible plasmids

It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors-pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl beta-d-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.     

An improved system for expressing pancreatic ribonuclease in Escherichia coli

An improved method for expressing and purifying bovine pancreatic ribonuclease from a synthetic gene using the lambda promoter controlled by a temperature-sensitive repressor is described. The procedure involves isolation in the presence of a refolding buffer containing oxidized and reduced glutathione, under conditions where RNase can refold, but where proteases presumably do not. Yields are approx. 2 mg purified protein per 1 ferment.     

An improved Escherichia coli donor strain for diparental mating

We present a new method for diparental mating with the outstanding advantage that counterselection of the Escherichia coli donor strain is not required. This improved method uses a new donor strain, E. coli ST18, a hemA deletion mutant defective in tetrapyrrole biosynthesis. The hemA mutation can be complemented by addition of 5-aminolevulinic acid. Therefore, counterselection is carried out only using standard media and growth conditions optimal for the recipient strain. Consequently, recipient strains are isolated in a significantly shorter period.     

Synthetic oligodeoxyribonucleotide probes for detecting heat-stable enterotoxin-producing Escherichia coli by DNA colony hybridization.

DNA colony hybridization was used to identify and enumerate enterotoxigenic Escherichia coli strains in foods. The cells were identified and enumerated by using synthetic polynucleotide probes for the heat-stable enterotoxin genes. These 22-base oligonucleotides, made from known nucleotide sequences of the genes for the heat-stable enterotoxins of human and porcine strains of E. coli, contain two mismatches between the two heat-stable enterotoxins. Colonies were replicated from agar medium onto paper filters and lysed with alkali followed by steam; probes were end labeled. After overnight hybridization at 40 degrees C and washing at 50 degrees C, autoradiograms were exposed at -70 degrees C. Results were consistent with suckling-mouse tests for heat-stable enterotoxins. A stronger signal was obtained on paper filters than on nitrocellulose filters. Enterotoxigenic E. coli cells were detected when mixed with a 1,000-fold excess of nonenterotoxigenic E. coli cells. This procedure appears to be more acceptable for routine testing than the use of cloned DNA fragments, labeling by nick translation, and lysing colonies on nitrocellulose filters.     

An improved statistical method for detecting heterotachy in nucleotide sequences

The principle of heterotachy states that the substitution rate of sites in a gene can change through time. In this article, we propose a powerful statistical test to detect sites that evolve according to the process of heterotachy. We apply this test to an alignment of 1289 eukaryotic rRNA molecules to 1) determine how widespread the phenomenon of heterotachy is in ribosomal RNA, 2) to test whether these heterotachous sites are nonrandomly distributed, that is, linked to secondary structure features of ribosomal RNA, and 3) to determine the impact of heterotachous sites on the bootstrap support of monophyletic groupings. Our study revealed that with 21 monophyletic taxa, approximately two-thirds of the sites in the considered set of sequences is heterotachous. Although the detected heterotachous sites do not appear bound to specific structural features of the small subunit rRNA, their presence is shown to have a large beneficial influence on the bootstrap support of monophyletic groups. Using extensive testing, we show that this may not be due to heterotachy itself but merely due to the increased substitution rate at the detected heterotachous sites.