Enzymatic multiplex DNA sequencing.

The problem of reading DNA sequence films has been reformulated using an easily implemented, multiplex version of enzymatic DNA sequencing. By utilizing a uniquely tagged primer for each base-specific sequencing reaction, the four reactions can be pooled and electrophoresed in a single lane. This approach has been previously proposed for use with fluorescently labelled probes (1), and is analogous to the principle used in four-dye fluorescence sequencing except that the signals are resolved following electrophoresis (2). After transfer to a nylon membrane, images are obtained separately for each of the four reactions by hybridization using oligonucleotide probes. The images can then be superimposed to reconstitute a complete sequence pattern. In this way the correction of gel distortion effects and accurate band registration are considerably simplified, as each of the four base-specific ladders require very similar corrections. The methods therefore provide the basis for a second generation of more accurate and reliable film reading programs, as well as being useful for conventional multiplex sequencing. Unlike the original multiplex protocol (3), the approach described is suitable for small projects, as multiple cloning vectors are not used. Although more than one vector can be utilized, only a library of fragments cloned into any single phage, phagemid or plasmid vector is actually required, together with a set of tagged oligonucleotide primers.     

Evidence for a new Escherichia coli protein resembling a lysyl-tRNA synthetase.

The amino acid sequence deduced from the nucleotide sequence of an open reading frame adjacent to the frdA gene of Escherichia coli shows 30.5% identity with the C terminus of Escherichia coli lysyl-tRNA synthetases. The three motifs characteristic of aminoacyl-tRNA synthetases of class 2 are recognizable within this sequence.     

Isotope partitioning in the adenosine 3',5'-monophosphate dependent protein kinase reaction indicates a steady-state random kinetic mechanism

Isotope partitioning beginning with the binary E.MgATP and E.N-acetyl-Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ser-peptide) complexes indicates that the kinetic mechanism for the adenosine 3',5'-monophosphate dependent protein kinase is steady-state random. A total of 100% of the initial radioactive E.MgATP complex is trapped as phospho-Ser-peptide at infinite Ser-peptide concentration at both low and high concentration of uncomplexed Mg2+, suggesting that the off-rate of MgATP from the E.MgATP.Ser-peptide complex is slow relative to the catalytic steps. Km for Ser-peptide in the trapping reaction decreases from 17 microM at low Mg2+ to 2 microM at high Mg2+, indicating that Mg2+ decreases the off-rate for MgATP from the E.MgATP complex. A total of 100% of the radioactive E.Ser-peptide complex is trapped as phospho-Ser-peptide at low Mg2+, but only 40% is trapped at high Mg2+ in the presence of an infinite concentration of MgATP, suggesting that the off-rate for Ser-peptide from the central complex is much less than catalysis at low but not at high Mg2+. In support of this finding, the Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly (Ala-peptide) increases from 0.27 mM at low Mg2+ to 2.4 mM at high Mg2+. No trapping was observed at either high or low Mg2+ for the E.MgADP complex up to a phospho-Ser-peptide concentration of 5 mM. Thus, it is likely that in the slow-reaction direction the kinetic mechanism is rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetics of the first wave of spermatogenesis in the newborn male mouse

A combination of autoradiography and air-dried techniques was used to calculate the duration of the major meiotic stages in the first wave of spermatogenesis in the newborn mouse. The data indicated that the entry into meiosis occurred asynchronously over 2 days, and the time required for each stage and the total cycle was constant. These time intervals were nearly identical with those estimated for adult animals in the present study and by other authors.     

Studies of myofibrillar ATPase in ragweed-sensitized canine pulmonary smooth muscle

The maximal shortening velocities of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized dogs were significantly higher than those of muscles from their littermate controls. Myofibrils of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized and control dogs were obtained with use of Triton X-100 homogenizing solution. The myofibrillar adenosinetriphosphatase (ATPase) activities of the sensitized tissues were significantly higher (P less than 0.05) than those of their respective controls.     

Pharmacological properties of canine intrapulmonary blood vessels

The contractile response of ring segments of large, medium, and small pulmonary arteries and veins of the dog to histamine, norepinephrine, and serotonin have been studied. The maximum contractile response to these drugs was normalized with respect to the maximal response obtained in stimulation with 127 mM K+. The small pulmonary artery was more reactive to histamine, norepinephrine, and serotonin when compared with large and medium pulmonary arteries. The medium and large pulmonary artery showed no difference in reactivity to histamine. However, the mean effective dose (ED50) values for these agonists among the different segments of pulmonary arteries showed no significant difference. The small and medium pulmonary veins demonstrated increased reactivity to histamine, but not norepinephrine and serotonin. The ED50 values also indicated that both small and medium veins were more sensitive to histamine when compared with the large pulmonary vein. The log concentration percent response curves for both small and medium pulmonary veins were displaced leftward (increased sensitivity) with respect to that for the large pulmonary vein. However, the reactivity and sensitivity to histamine between medium and small pulmonary veins were no different. The reactivity and sensitivity of different segments of pulmonary veins to norepinephrine and serotonin showed no significant differences among them. We conclude that histamine and other vasoactive substances, which are directly or indirectly related to mast cell degranulation, exert pharmacological effects on the pulmonary vasculature which possesses differential responsiveness at various levels of the vascular tree.     

油菜细胞质雄性不育系叶绿体DNA特异片段的分子克隆

采用高离子浓度缓冲液法分别提取油菜不育系及保持系的叶绿体DNA。经Sepharcse 4B柱层析纯化后,得到具有较高纯度的叶绿体DNA样品。将其分别用Eco RI、Bam HI、HimdHI、PstI和XhoI 5种限制性内切酶酶解,得到5种限制性内切酶图谱。其中除PstI图谱外,其它4种酶谱均显示出明显的两系间叶绿体DNA结构差异。以pBR 322为载体,分别克隆了不育系Bam HI图谱上的3个特异片段。得到的3个克隆,经克隆杂交及电泳分析后,证实分别带有上述3个目的片段。这些特异片段的特性及其与花粉育性的关系尚在研究中。     

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.     

Resonance Raman spectra of the heme in leghemoglobin. Evidence for the absence of ruffling and the influence of the vinyl groups

Resonance Raman spectra of deoxy and carbonmonoxy leghemoglobin (Lb) are compared to the corresponding forms of human adult hemoglobin (HbA). It is found that the heme "core size" indicator line has nearly the same frequency for the two deoxyhemoglobins and the pi-electron density-sensitive line also falls at the same frequency. However, several other modes occur at very different frequencies in the spectra of the two proteins. From an examination of the spectrum of an HbA derivative in which the beta-carbon atoms of the heme vinyl groups were deuterated, it appears that the major differences between deoxy-HbA and -Lb may result from conformational changes in the vinyl groups. No evidence for the suggested ruffling (Irwin, M. J., Armstrong, R. S., and Wright, P. E. (1981) FEBS Lett. 133, 239-243) in deoxy-Lb was found. The spectra of carbonmonoxy-Lb and -HbA were also found to be very different. As in the deoxy case, some of these frequency differences could be attributed to vinyl group conformational differences. However, from the large difference in the pi-electron density-sensitive line, it appears that the vinyl pi-conjugation into the porphyrin in Lb(CO) may be different than it is in HbA(CO). The vinyl conformational differences may be a consequence of the looser heme pocket in Lb than in HbA. The difference in pi-conjugation could make a significant contribution to the difference in ligand binding affinity for these two globins.