Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

DNA启动子的计算机识别

本文介绍了两种预测DNA顺序上的启动子位置的计算机识别方法,方法1是基于启动子部位单核苷酸的分布不均一性,方法2是基于启动子部位二核苷酸的分布不均一性。分别用这两种方法推测了质粒pBR322DNA上启动子的位置,从而验证了化学实验的结果。     

Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains

The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours. On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA ^–] bacteria is similar. We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom ^– in both relA ^– and relA ⁺ strains during amino acid starvation. Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom ^–, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria. A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Efficient transformation of a Yersinia enterocolitica strain (serotype O:3) by pBR322 DNA

Abstract The Yersinia enterocolitica strain MS201 (serotype O:3) was transformed by pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from the transformed Y. enterocolitica is able to transform plasmid-free MS201 at a significantly higher frequency, suggesting the existence of a restriction-modification system in MS201.     

Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone

The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous.     

Survival and transfer in the human gut of poorly mobilizable (pBR322) and of transferable plasmids from the same carrier E. coli

We examined the survival of a host Escherichia coli K-12 bacterium containing two transferable plasmids (pLM2, pSL222-4) and one poorly mobilizable plasmid (pBR322), and the transfer of these three plasmids to endogenous bacteria in the human intestinal tract. The survival of this plasmid-carrying host organism in four human volunteers was 3.5 to 6 days at recovery rates of 10^?1 to 10^?4. This finding was similar to our previous survival data on the same organism bearing a single plasmid. The K-12 strain appeared to be under a strong selective disadvantage in the human gut, since, even when bearing a tetracycline-resistant plasmid, its titer did not increase despite the administration of tetracycline. Studies of transferability showed that, while the transfer-depressed incFII plasmid pSL222-4 transferred at a frequency of 10^?1 in culture, its transfer in the human gut was much less frequent. The number of new recipients per donor cell ingested was about 10^?5, which included new recipients arising by multiplication. The recovery of pSL222-4 transcipients was enhanced by the administration of tetracycline on day 6. Neither the transfer-repressed, broad host range incP plasmid pLM2, nor the plasmid pBR322, could be detected in any endogenous host bacteria. Using the transfer and mobilization frequencies obtained in culture and the number of new recipients of pSL222-4 in the intestinal tract, we estimated that any in vivo mobilization of pBR322 to a new recipient could not occur at a frequency higher than 10^?12.     

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apoVLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellinin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.