Plasmid construction by homologous recombination in yeast

We describe a convenient method for constructing new plasmids that relies on interchanging parts of plasmids by homologous recombination in Saccharomyces cerevisiae. A circular recombinant plasmid of a desired structure is regenerated after transformation of yeast with a linearized plasmid and a DNA restriction fragment containing appropriate homology to serve as a substrate for recombinational repair. The free ends of the input DNA molecules need not be homologous in order for efficient recombination between internal homologous regions to occur. The method is particularly useful for incorporating into or removing from plasmids selectable markers, centromere or replication elements, or particular alleles of a gene of interest. Plasmids constructed in yeast can subsequently be recovered in an Escherichia coli host. Using this method, we have constructed an extended series of new yeast centromere, episomal and replicating (YCp, YEp, and YRp) plasmids containing, in various combinations, the selectable yeast markers LEU2, HIS3, LYS2, URA3 and TRP1.     

戴氏狼鳍鱼(Lycoptera davidi)的重新观察

本文主要对戴氏狼鳍鱼 (Lycoptera davidi) 骨骼系统的特征进行了补充和订正.在此基础上,对狼鳍鱼属 (Lycoptera) 的特征及有关狼鳍鱼种的分类问题作了初步探讨.     

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants containing different point mutations in the HXK2 gene were used to study the relationship between phosphorylation by hexokinase II and glucose repression in yeast cells. Mutants showing different levels of hexokinase activity were examined for the degree of glucose repression as indicated by the levels of invertase activity. The levels of hexokinase activity and invertase activity showed a strong inverse correlation, with a few exceptions attributable to very unstable hexokinase II proteins. The in vivo hexokinase II activity was determined by measuring growth rates, using fructose as a carbon source. This in vivo hexokinase II activity was similarly inversely correlated with invertase activity. Several hxk2 alleles were transferred to multicopy plasmids to study the effects of increasing the amounts of mutant proteins. The cells that contained the multicopy plasmids exhibited less invertase and more hexokinase activity, further strengthening the correlation. These results strongly support the hypothesis that the phosphorylation activity of hexokinase II is correlated with glucose repression.     

Microcalorimetric measurements of tissue cells in vitro

A culture flask was designed for the microcalorimetric measurements of tissue cells by an MS 80 standard calvet microcalorimeter. Tissue cells cultured in this flask behaved in the same manner as in the common culture flask used in cytobiological studies. The thermograms of human adenocarcinoma gastric cells (SGc 7901) and HeLa cells were obtained. The heat output power of SGc 7901 cells continuously increased for 70 h with an initial cell number of 3.0 X 10(5). The thermogram was reproducible under strictly controlled conditions. The relationship between the heat output power and the number of SGc 7901 cells within 48 h was obtained. The heat output power was 40 pW/cell to 49 pW/cell when the cell number was in the range 4.5 X 10(5) to 10.4 X 10(5). It was 62.3 +/- 2.9 pW/cell for HeLa cells when the cell number was 6 X 10(5).     

Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats

With the techniques of specific radioimmunoassay and gel filtration it was found that CGRP was distributed in various tissues of normotensive (WKY) and spontaneously hypertensive rats (SHR) with the highest concentration in the lumbar spinal cord (1197 +/- 94.8 pg/mg tissue) and the lowest in the auricle (15.0 +/- 2.1 pg/mg tissue). In comparison with WKY, CGRP concentration in the plasma was decreased and in the abdominal aorta and hypothalamus was increased in SHR. Gel filtration revealed only one major CGRP molecular form in the tissues. In addition, CGRP reduced the mean arterial pressure (MAP) in SHR in a dose-dependent manner. These data suggest that CGRP may play an important role in the pathogenesis of hypertension and its possible therapy.     

大壁虎的染色体及减数分裂联会复合体的研究

大壁虎(Gekko gecko)的染色体数目为2n=38,核型由2对中着丝粒(Nos.1.4.)、3对亚中着丝粒(Nos.2.3.5)及14对端着丝粒和亚端着丝粒(Nos.6—19)染色体组成。一对核仁组织者(NOR_s),位于第7对端着丝粒染色体的末端。同时,本文还对大壁虎的减数分裂以及联会复合体(S.C)的结构和组型,进行了详细的观察和分析。     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

Brown adipose tissue, liver, and diet-induced thermogenesis in cafeteria diet-fed rats

Diet-induced thermogenesis (DIT) in young rats overeating a "cafeteria" (CAF) diet of palatable human foods is characterized by a chronic, propranolol-inhibitable elevation in resting metabolic rate (VO2) and is associated with various changes in brown adipose tissue (BAT) that have been taken as evidence for BAT as the effector of DIT. But direct evidence for participation of BAT in DIT has been lacking. By employing a nonocclusive cannula to sample the venous effluent of interscapular BAT (IBAT) for analysis of its O2 content and measuring tissue blood flow with microspheres, we accomplished direct determination (Fick principle) of the O2 consumption of BAT in conscious CAF rats. In comparison with normophagic controls fed chow, the CAF rats exhibited a 43% increase in metabolizable energy intake, reduced food efficiency, a 22% elevation in resting VO2 at 28 degrees C (thermoneutrality) or 24 degrees C (housing temperature), and characteristic changes in the properties of their BAT (e.g., increased mass, protein content and mitochondrial GDP binding). They also exhibited the greater metabolic response to exogenous noradrenaline characteristic of CAF rats and the near elimination by propranolol of their elevation in VO2. By the criterion of their elevated VO2, the CAF rats were exhibiting DIT at the time of the measurements of BAT blood flow and blood O2 levels. However, BAT O2 consumption was found to be no greater in the CAF rats than in the controls at either 28 or 24 degrees C. At 28 degrees C it accounted for less than 1% of whole body VO2; at 24 degrees C it increased to about 10% of overall VO2 in both diet groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.     

Biosynthesis of fusarochromanone and its monoacetyl derivative by Fusarium equiseti.

One fluorescent compound previously named TDP-2 was isolated and purified from a rice culture of Fusarium equiseti (Alaska 2-2). Mass spectral and nuclear magnetic resonance data indicated that it is a C-3'-N-acetyl derivative of fusarochromanone, a newly discovered mycotoxin. Time course studies of synthesis of these two compounds on autoclaved rice and Czapek-Dox medium enriched with soybean peptone indicated that fusarochromanone was converted to TDP-2 in the cultures. A high concentration of peptone in the liquid medium may stimulate both fusarochromanone synthesis and its conversion to TDP-2.