原子力显微镜研究不同温度下质粒DNA结构变化

采用原子力显微镜（ＡＦＭ）在无水乙醇中成象了ｐＢＲ３２２质粒ＤＮＡ。在不同温度范围加热云母表面的ＤＮＡ溶液,并在相应温度下干燥使ＤＮＡ充分展开地固定在云母基底上。ＡＦＭ观察结果表明：在３０～４０℃,质粒ＤＮＡ主要是超螺旋和环状分子;在４０～５０℃,环状分子开环形成线形分子;　在７０～８０℃,这些线形分子全部变性,解链形成无规线团。我们通过测定ｐＢＲ３２２质粒ＤＮＡ的熔融曲线,发现了两个突变点,对应于ＡＦＭ所观察到的质粒ＤＮＡ在加热过程中由环状变成线形、双螺旋解开成单链的两个结构变化过程。这是首次通过ＡＦＭ直接观察到质粒ＤＮＡ在不同温度下的结构变化。     

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。     

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.     

Restriction endonuclease BamHI cleaves bacteriophage Mu DNA within cistrons E and F

We have cleaved phage Mu DNA with restriction endonucleases EcoRI and BamHI and have cloned three specific DNA fragments from the middle of the Mu genome into vector plasmid pBR322. By marker rescue experiments, we have determined that the two BamHI cleavage sites in Mu DNA occur within cistrons E and F.     

Expression of the yeast galactokinase gene in Escherichia coli.

In Saccharomyces cerevisiae the genes for three of the enzymes involved in galactose metabolism are tightly linked near the centromere of chromosome II (Douglas and Hawthorne, 1964). However, the molecular mechanisms which control the expression of these genes are not well understood. A DNA fragment containing at least one of these yeast genes, the galactokinase gene (gal1), has been joined to the bacterial plasmid pBR322 and maintained in an Escherichia coli strain that carries a deletion in its own galactokinase gene, galK. The presence of the yeast gene was demonstrated by (i) complementation of the E. coli galactokinase deletion, (ii) by hybridization of the cloned DNA fragment to restriction enzyme digests of total yeast DNA and (iii) by assaying for yeast galactokinase activity in bacterial cell extracts. The yeast DNA fragment is 4700 base pairs long, and enables the host E. coli K-12 strain to grow in minimal medium containing galactose as the sole carbon source with a generation time of 14.3 h. The yeast galactokinase activity in the bacterial extracts is 0.7% of the bacterial galactokinase activity found in wild-type E. coli fully induced with fucose.     

Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments.

A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a mating-type-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.     

Methylation sensitivity of the restriction enzymes FnuDII and AccII

The restriction enzymes FnuDII and AccII are isoschizomers of the DNA sequence 5-CGCG-3. We have determined that 5-methylcytidine at either cytidine position in this recognition sequence inhibits DNA cleavage by FnuDII and AccII. A third isoschizomer, ThaI was previously shown to exhibit an identical methylation sensitivity. It is remarkable that 3 restriction enzymes derived from diverse microbiological sources exhibit this identical methylation sensitivity.Abbreviations bp base pair(s) - m_c 5-methylcytidine - Tris-borate buffer 89 mM Tris-base, 89 mM boric acid, 2.5 mM EDTA, pH 8.3     

Rat immunoglobulin delta heavy chain gene: nucleotide sequence derived from cloned cDNA

Rat immunoglobulin δ heavy-chain mRNA has been isolated. RNA blot analysis revealed that this mRNA with a length of 1.8 kb encodes for the secreted form of IgD. The corresponding cDNA was cloned in plasmid pBR322 and its sequence was determined. The hybrid plasmid contains a 775-bp insert comprising a partial Cδ₁ sequence and complete CδH, Cδ3, CδDC and 3' untranslated sequences. Rat and mouse IgD amino acid sequences show striking homology in Cδ3 and CδDC regions.     

DNA binding proteins of rat thigh muscle: Purification and characterization of an endonuclease

Two major DNA binding proteins of molecular weights 34,000 and 38,000 have been identified in the 30,000 g supernatant (S-30) fraction of rat thigh muscle extracts. The presence of 38 KD DNA binding protein in the muscle S-30 could be demonstrated only if Triton X-100 treated extracts were used for Afinity chromatography suggesting that this protein may be a membrane associated DNA binding protein. The 38 KD DNA binding protein differed from the 34 KD DNA binding protein also in its chromatographic behaviour in DE-52 columns in which the 38 KD protein was retained, while the 34 KD protein came out in the flow-through in an electrophoretically pure form. The 34 KD DNA binding protein can also be purified by precipitation with MgCl₂. Incubation of 0 15 M NaCl eluates (containing the 38 KD and/or 34 KD DNA binding protein) in the presence of 100 mM Mg²⁺ resulted in the specific precipitation of the 34 KD protein. Prolonged incubation (30 days) of the 0.15 M NaCl eluates containing the two DNA binding proteins at 4°C led to the preferential degradation of the 34 KD DNA binding protein. Nitrocellulose filter binding assays indicated selective binding of purified 34 KD protein to ss DNA. Purified 34 KD DNA binding protein cleaved pBR 322 supercoiled DNA, and electrophoresis of the cleavage products in agarose gels revealed a major DNA band corresponding to the circular form of DNA.     

Restriction map, partial cloning and localization of 9S and 12S kinetoplast RNA genes on the maxicircle component of the kinetoplast DNA of Leishmania tarentolae.

We have constructed a restriction map of the maxicircle component of the kinetoplast DNA of Leishmania tarentolae for the enzymes EcoRI, Bam HI, HaeIII, HpaII, SalI, BglII and HindIII. The 9 and 12S kinetoplast RNAs were localized on this map. Two fragments of this maxicircle molecule were cloned in the bacterial plasmid, pBR322, including a 4.4 . 10(6) dalton EcoRI/BamHI fragment which contains the 9 and 12S RNA genes.     

A thermoinducible lambda phage-ColE1 plasmid chimera for the overproduction of gene products from cloned DNA segments.

Two segments of lambda have been cloned into the multicopy plasmid pBR322. One extends from N through cII (NcII segment, from 71.3 to 81.0% on the physical map) and the other from N through P (NOP segment, from 71.3 to 86.5% on the physical map). Cells carrying these recombinant plasmids express lambda immunity (cIts) and Rex function. In addition, they decrease the efficiency of plating at 32 degrees C of lambdavir and lambdaimm434, but not that of lambdaimm21. Recombinant plasmids with lambdaNOP segments (pKC14, pKC16) differ from recombinant plasmid with labmdaNcII segment (pKC10) in two respects: (i) strains carrying pKC14 or pKC16 are killed at 42 degrees C, and (ii) these strains are thermally inducible for plasmid DNA synthesis, resulting in increase of plasmid copy number from an uninduced level of 50 to more than 130 per chromosome. It was suggested that both these differences are related to functions contained in the lambda DNA segment extending from 81.0 to 86.5%. The usefulness of plasmid pKC16 for overproduction of gene products from cloned DNA segments was demonstrated by cloning the E. coli exonuclease III gene (xth) in pKC16. Thermal induction of this xth plasmid (pSGr) results in a 125-fold increase in exonuclease III activity over that of a control strain lacking the xth gene insert. The extent of exonuclease III overproduction obtained by cloning xth gene in a lambda vector was similar to that obtained with pSGR3.     

Studies on the organization of the α-satellite DNA from African green monkey cells using restriction nucleases and molecular cloning

α-Satellite DNA from African green monkey cells was analysed with restriction nucleases in some detail confirming and complementing our earlier results. With EcoRI and HaeIII (or BsuRI isoschizomer), about 25 and 10%, respectively, of the satellite DNA were cleaved into a series of fragments of the 172 bp repeat length and multiples thereof. To allow studies with fragments of homogeneous sequence unit length, HindIII fragments were covalently joined with the plasmid pBR313. After transformation 19 clones were obtained, containing up to three monomer fragments. Nine of the clones were characterized by digestion with EcoRI. Three of these had cleavage sites for this nuclease in the satellite DNA portion. In the six clones tested with HaeIII no cleavage site was detected in the cloned DNA. The results are discussed in relation to the nucleotide sequence data recently published by Rosenberg et al. (1978) and in the context of random and nonrandom processes in satellite DNA evolution.     

The nucleotide sequence of cDNA coding for preproinsulin from the primate Macaca fascicularis

DNA complementary to preproinsulin messenger RNA from the primate Macaca fascicularis has been cloned into the PstI endonuclease site of the plasmid pBR322. One clone contains the entire preproinsulin coding region as well as 59 nucleotides of the 5'-untranslated region. The results predict an amino acid sequence for the Macaca fascicularis preproinsulin and establish for the first time that the primary structures of human and primate insulins are identical. The two amino acid exchanges between human and primate preproinsulins are restricted to the pre- and the C-peptide, respectively.     

Construction of restriction enzyme fragment libraries containing DNA from human adenovirus types 2 and 5

Restriction-fragment libraries containing adenovirus type 2 (Ad2) DNA have been constructed, using the pBR322 plasmid (Bolivar et al., 1977) as a vector. Clones have been isolated which contain all the HindIII fragments of Ad2 DNA except the terminal G- and K-fragments inserted into the HindIII cleavage site of the vector. All the 13 SmaI-fragments of Ad2 DNA were separately inserted into the PstI site of the pBR322 vector after addition of homopolymeric poly(dG) tails to the fragments and poly(dC) tails to the linearized plasmid. Two large fragments of adenovirus type 5 (AD5) DNA, located between map positions 17.0 and 59.5 and between map positions 59.5 and 97.3, respectively, were cloned using bacteriophage lambda as a vector. All clones, which are described in the present report, are available upon request.     

Cloning and expression in Escherichia coli of a species-specific Chlamydia trachomatis outer membrane antigen

Abstract A gene library of Chlamydia trachomatis serovar L₂ (strain 434) was constructed in Escherichia coli using plasmid pBR322. Amongst 200 recombinants we have identified and characterized a recombinant E. coli that expresses a protein antigen of M _r 74 000 similar in size to an outer membrane antigen produced by elementary bodies of C. trachomatis . Immunologically, the molecule synthesised by E. coli has the same specificity as the protein encoded by serovar L₂. A 1.8 kb DNA fragment from the recombinant insert, used as a hybridization probe, confirmed the species specificity of this clone at the gene level.