Specificity in the formation of delta tra F-prime plasmids.

Twenty-three independent delta tra F-prime plasmids from three different Escherichia coli K-12 sublines were isolated from Hfr strains whose points of origin coincided with the IS3 element alpha 3 beta 3 or alpha 4 beta 4 in the lac-purE region of the E. coli chromosome. Electrophoretic analysis of plasmid deoxyribonucleic acid digested with EcoRI and hybridization analysis of plasmid deoxyribonucleic acid digested with BglII revealed that at least 14 of these plasmids were formed by processes involving specific bacterial and F loci. Two of the specific bacterial loci involved in delta tra F-prime formation were located at approximately 3.3 and 11.7 min on the E. coli chromosomal map. Two of the delta tra F-prime plasmids contained bacterial deoxyribonucleic acid with circularization endpoints that mapped very near the termini of the IS2 element that is normally located between lac and proC.     

Formation of delta tra F' plasmids: specific recombination at oriT

Delta tra F' plasmids can be isolated from matings between Hfr donors and recA- recipients, with selection for transfer of proximal chromosomal genes. Previous experiments indicate that F DNA from the neighborhood of the transfer origin up to the proximal junction with the chromosomal DNA is present on these plasmids, together with chromosomal segments, some of which belong to distinct size classes. We have sequenced across the novel joints contained in five delta tra FproA+ plasmids and in five delta tra FpurE+ plasmids, and we have compared these with the F sequence near oriT and with a chromosomal site near purE. The previously reported specificity in formation of some of these classes is confirmed at the nucleotide sequence level. The F DNA in nine of these novel joints extended beyond the nicking sites identified by others in lambda oriT+ bacteriophages up to a position between two sequenced oriT- mutations. Small plasmids containing these novel joints are mobilized in trans by pOX38 at frequencies less than 5 X 10(-7) times the mobilization frequencies for similar plasmids that contain oriT. The relations of these findings to the location of the nicking site at oriT are discussed.     

Systems regulating the tra genes of derepressed F-like plasmids

A study was made of the ability of reference plasmids of the 6 known Fin-groups to inhibit the functions of transfer genes (tra-genes) of the 4 derepressed F-like plasmids (pAP22-2, pAP38, pAP43, pAP53). It was shown that unlike the derepressed Flac plasmid, the conjugation transfer of pAP38 and pAP53 plasmids was inhibited only by, the FinV plasmid, whereas pAP22-2 plasmids by Fin V and Fin V plasmids. The formation of donor-specific pili in case of pAP38 plasmid was inhibited by Fin Q, Fin U and Fin V plasmids, in case of pAP43 plasmid by Fin U Fin V and Fin W plasmids.     

Pili determined by the tra genes of F-like plasmids

The study of structural and functional features of plasmid-specific pili synthetized by E. coli cells under control of 27 F-like plasmids was performed. All the plasmids determined the pili of "flexible" type which were classified into 3 groups on the basis of difference in cell sensitivity to pili-specific phages f1, f2, Q. The possible role of transposons in the variation of pili functional properties was supposed.     

Heteroduplex analysis of the RNA of clone 3 Moloney murine sarcoma virus.

Heteroduplex analysis of the RNA isolated from purified virions of clone 3 Moloney murine sarcoma virus (M-MSV) hybridized to cDNA's from Moloney murine leukemia virus (M-MLV) and clone 124 M-MSV shows that the main physical component of clone 3 RNA is missing all or most of the 1.5-kilobase (kb) clone 124 M-MSV specific sequence denoted beta s (S. Hu et al. Cell 10:469--477, 1977). This sequence is either deleted in clone 3 RNA or substituted by a very short (0.3-kilobase) sequence. In other respects, clone 3 and clone 124 RNAs show the same heteroduplex structure relative to M-MLV. Since beta s is believed to contain the src gene(s) of clone 124 RNA, this result leaves as an unresolved question the nature of the src gene(s) of the clone 3 M-MSV RNA complex.     

Specificity in formation of type II F'' plasmids.

Eight new F' plasmids derived from Hfr strains in which F is integrated at the chromosomal element alpha 3 beta 3 have been isolated and subjected to restriction enzyme, hybridization, and electron microscope heteroduplex analysis. Plasmids carrying extensive amounts of bacterial deoxyribonucleic acid were produced even though they were obtained by selection for transfer of lac, which is closely linked to F in the parental Hfr strains. Seven plasmids were type II Flac+ proC+ purE+ plasmids, and one was a type I Flac+ proC+ plasmid. Five of the Flac+ proC+ purE+ plasmids contain approximately 284 kilobases of bacterial deoxyribonucleic acid, which is identical for all five within the resolution of the restriction enzyme analysis. Theses results indicate that type II F' plasmids are the predominant tra+ F' type from this region of the Escherichia coli K-12 chromosome and that the recombination events leading to formation of these plasmids exhibit site specificity.     

Reduction-driven polypeptide folding by the delta Tt mechanism.

Poly(Gly-Val-Gly-Val-Pro), i.e., poly(GVGVP), exhibits composition and solute dependence of Tt, the temperature of the inverse temperature transition at which hydrophobic folding and assembly occur on raising the temperature. Importantly, a means whereby the value of Tt is lowered from above to below the working temperature becomes an isothermal means of driving folding and assembly, i.e., of achieving free energy transduction. Using poly[0.73(GVGVP),0.27(GK[NMeN]GVP)] where [NMeN] indicates N-methyl nicotinamide attached to the epsilon-NH2 of the Lys(K) residue, chemical and electrochemical reductions are found to remarkably lower the value of Tt; reduction can drive hydrophobic folding and assembly as effectively as decreasing ionization. Changing the redox state of a protein becomes yet another means of achieving free energy transduction by the delta Tt mechanism.     

Ribozymes of the hepatitis delta virus: recent findings on their structure, mechanism of catalysis and possible applications.

Although the delta ribozymes have been studied for more than ten years the most important information concerning their structure and mechanism of catalysis were only obtained very recently. The crystal structure of the genomic delta ribozyme turns out to be an excellent example of the extraordinary properties of RNA molecules to fold into uniquely compact structures. Details of the X-ray structure have greatly stimulated further studies on the folding of the ribozymes into functionally active molecules as well as on the mechanism of RNA catalysis. The ability of the delta ribozymes to carry out general acid-base catalysis by nucleotide side chains has been assumed in two proposed mechanisms of self-cleavage. Recently, considerable progress has been also made in characterizing the catalytic properties of trans-acting ribozyme variants that are potentially attractive tools in the strategy of directed RNA degradation.     

Small-scale techniques for the analysis of recombinant plasmids.

Using the cloning of part of the T-DNA of pTiC58 from Agrobacterium tumefaciens as an example, techniques are described which enable recombinant plasmids to be mapped and used as hybridization probes. In all cases the starting material is a colony of cells grown on an agar plate which is then subjected to lysis by lysozyme and Triton X-100 in volumes of the order of 300 microliters thus eliminating the need for handling and centrifuging liquid cultures under restrictive containment conditions.     

热带森林的低密度种及其形成及维持机制

低密度种指在热带森林中存在的成年个体密度很低的物种 ,是热带森林中树种的主要存在形式。对低密度种的认识和保护是热带森林生物多样性保育的关键之一。与稀有种相比 ,低密度种的含义更为广阔 ,它还包括那些密度低、分布范围广和绝对个体数量大的物种。低密度种形成的主要原因是母树附近的幼苗和种子的存活率较低 ,密度制约和有限更新也是低密度种形成的重要原因。维持低密度种最重要的外部原因是昆虫较强的飞行能力和传粉能力 ,而其最重要的内部原因是低密度种的生殖特性。     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.