Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.     

Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase

We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).     

Cathepsin B secretion by rabbit articular chondrocytes: modulation by cycloheximide and glycosaminoglycans

Rabbit articular chondrocytes in monolayer culture are modulated away from their differentiated state and undergo morphological and biochemical changes. One of the characteristics of the modulated state is an abnormally high production of the cysteine endopeptidase cathepsin B. Addition to chondrocyte cultures of the protein biosynthesis inhibitor, cycloheximide, resulted in a concentration-dependent reduction of cathepsin B secretion, which was fully restored after removal of cycloheximide. Glycosaminoglycans added to the culture medium of modulated chondrocytes partially reduced the rate of secretion of cathepsin B, this effect being dependent on their structure, the degree of sulfation, and concentration. The age of the chondrocytes and the duration of the treatment also influenced this response. The switching off of cathepsin B release was apparently best favored by a high concentration of negatively charged sulfate groups attached to a polymeric glycosaminoglycan chain; this simulates the natural environment of the chondrocytes in articular cartilage.     

Sex determination in the dioecious Melandrium

Summary Melandrium album (2n=24), a dioecious species with heteromorphic sex chromosomes (XY, males and XX, females), has a strong genetic commitment for sex determination. We report here a procedure for obtaining haploid plants from cultured anthers and show that genotype, pollen stage, cold treatment and certain culture media components are essential for a reproducible yield of embryos. Our procedure increased the number of responsive anthers and not the number of responsive microspores per anther. Most likely, our experimental system allows the recovery of competent microspores, and this on a medium containing either an auxin or a cytokinin. All of the 36 anther-derived plants tested expressed a female phenotypic sex instead of the theoretical one male one female ratio. When analysed cytologically, the plants exhibited the corresponding female genetic sex (one or two X chromosomes).     

The relationship between RNA catalytic processes

Proposals that an RNA-based genetic system preceeded DNA, stem from the ability of RNA to store genetic information and to promote simple catalysis. However, to be a valid basis for the RNA world, RNA catalysis must demonstrate or be related to intrinsic chemical properties which could have existed in primordial times. We analyze this question by first classifying RNA catalysis and related processes according to their mechanism. We define: (A) thedisjunct nucleophile class which leads to 5-phosphates. These include Group I and II intron splicing, nuclear mRNA splicing and RNase P reactions. Although Group I introns and its excision mechanism is likely to have existed in primordial times, present-day examples have arisen independently in different phyla much more recently. Comparative methodology indicates that RNase P catalysis originated before the divergence of the major kingdoms. In addition, alldisjunct nucleophile reactions can be interrelated by a proposed mechanism involving a distant 2-OH nucleophile. (B) theconjunct nucleophile class leading to 3-phosphates. This class is composed of self-cleaving RNAs found in plant viruses and the newt. We propose that tRNA splicing is related to this mechanism rather than the previous one. The presence of introns in tRNA genes of eukaryotes and archaebacteria supports the idea that tRNA splicing predates the divergence of these cell types.     

湖北贝母单鳞片砂培繁殖研究

本文针对湖北贝母生产中存在繁殖系数低的问题,研究了单鳞片砂培繁殖对提高鳞茎繁殖率的效果和原理。试验结果表明:1.单鳞片繁殖率为对照种鳞茎的5—9倍,2.低温(2—10℃)预处理4—8周和暗条件培养,能有效地提高子球形成率,促使子球迅速长大,3.植物激素(6-BA、KT、2,4-D)处理,有利于促进鳞片不定芽原基的分化,繁殖率为种茎繁殖的9—11倍;4.单鳞片繁殖的小鳞茎主要发生在鳞片基部的茎盘上,还可发生在鳞片的远轴面上,但不发生在近轴面。     

一种纯化细菌有机磷水解酶的简便方法

Cibacron blue T_3GA与溴化氰活化的Sepharose 4B偶联后,产生一种能有效地分离有机磷水解酶的吸附剂。用0.15mol/L MgCl_2溶液从黄杆菌P3—2细胞抽提出的粗酶液通过柱层析分离,即可得到纯化8倍、酶活性回收率为269.4％的纯酶制品。该酶制品用凝胶电泳测是均一的。     

Domains required for nucleic acid binding activities in chloroplast ribonucleoproteins.

Five ribonucleoproteins (or RNA-binding proteins) from tobacco chloroplasts have been identified to date; each of these contains an acidic N-terminal domain (24-64 amino acids) and two conserved RNA-binding domains (82-83 amino acids). All five ribonucleoproteins can bind to ssDNA and dsDNA but show high specificity for poly(G) and poly(U). Here we present the nucleic acid binding activity of each domain using a series of deletion mutant proteins made in vitro from the chloroplast 29 kDa ribonucleoproteins. The acidic domain does not have a positive effect on binding activities and proteins lacking this domain show higher affinities for nucleic acids than the wild-type proteins. Mutant proteins containing single RNA-binding domains can bind to poly(G) and poly(U), though with lower affinities than proteins containing two RNA-binding domains. The spacer region (11-37 amino acids) between the two RNA-binding domains does not interact with poly(G) or poly(U) by itself, but is required for the additive activity of the two RNA-binding domains. Proteins consisting of two RNA-binding domains but lacking the spacer have the same activity as those containing only one RNA-binding domain. Possible roles for each domain in chloroplast ribonucleoproteins are discussed.     

鹅掌楸种子和胚胎发育的研究

应用控制授粉、软 X-射线法、常规石蜡制片法和荧光检测等手段,研究了鹅掌楸(Lirio-dendron chinense(Hemsl.)Sarg.胚胎发育和控制授粉与结籽率的相关性。控制授粉后2小时花粉萌发,6小时萌发率最高,柱头可授期持续30小时左右。花粉管借助于柱头毛之间的分泌物进入柱头沟,经花柱沟、珠孔塞和珠心冠原进入胚囊,行珠孔受精。授粉后2周,胚乳为2至3细胞厚的狭组织;第6周,胚乳充满胚囊腔,珠心随之解体殆尽;第7到8周,球形胚、心形胚发生;第14到16周,子叶形成;第22周种子或熟,胚乳丰富。单株自然授粉结籽率不足1%。控制授粉后,单个聚合果的最高结籽率可达39%,9个聚合果的平均结籽率为17.7%。     

Differentiation of somatic embryos from protoplasts isolated from embryogenic suspension cultures of Abies alba L.

Embryogenic suspension cultures of Abies alba were established using an embryogenic suspensor mass culture originating from the zygotic embryo in immature seed explants (Schuller et al. 1989). Protoplasts were isolated from the suspension material. The protoplasts were immobilized in alginate layers in order to follow the development of single protoplasts. During the first days of protoplast culture a modified Kao and Michayluk (1975) medium proved to be necessary for subsequent divisions. The formation of proembryos succeeded within 2–3 weeks when subcultured with a modified Schenk and Hildebrandt (1972) liquid medium. Light, enhanced sugar concentration, and the addition of abscisic acid led to the formation of slightly green torpedo-shaped somatic embryos after 6–8 weeks from protoplast isolation.Abbreviations ABA abscisic acid - BAP N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ESM Embryonal suspensor mass (Gupta and Durzan 1986) - KM Kao and Michayluk (1975) - LP (von Arnold and Eriksson 1977) - MES 2-(N-morpholino)ethane-sulfonic acid - NAA 1-naphthalene-acetic acid (sodium salt) - PVP polyvinylpyrrolidone - SH Schenk and Hildebrandt (1972) - Tween 80 polyoxyethylene-sorbitan-monooleate