一种粗糙脉孢霉基因组DNA的快速制备方法

粗糙脉孢霉基因组ＤＮＡ的制备方法一般很费工费时。ＷｅｎｄｌａｎｄＪＡ等人发展了一种丝状真菌的ＤＮＡ提取方法 ,应用在裂褶菌取得了良好的效果[1] 。本文基于该方法制备粗糙脉孢霉基因组ＤＮＡ也取得了成功 ,应用ＰＣＲ从基因组扩增出了一个与无机焦磷酸酶有同源性的基因。1　材料与方法1 1　菌种 :粗糙脉孢霉 (Ｎｅｕｒｏｓｐｏｒａｃｒａｓｓａ)菌种 490 7ｐｒｄ - 4 ,ｂｄＡ ,来自ＦｕｎｇａｌＧｅｎｅｔｉｃｓｓｔｏｃｋｃｅｎｔｅｒ,ＵｎｉｖｅｒｓｉｔｙｏｆＫａｎｓａｓＭｅｄｉｃａｌＣｅｎｔｅｒ,Ｋａｎｓａｓ ,ＵＳＡ。1 2…     

Specific interactions of distamycin A and its analogs with (A-T) rich and (G-C) rich duplex regions of DNA and deoxypolynucleotides.

The specific interaction of distamycin A and analogs with DNA's and synthetic deoxypolynucleotide duplexes were studied in detail by means of circular dichroism and the data were analyzed together with viscosity results of several natural DNA's. At low ligand to nucleotide ratio the previously reported specific binding to (A-T) pairs of DNA is verified by a highly favoured interaction with (A-T)-enriched segments of distamycins containing four and five methylpyrrole carboxamide units. At higher distamycin concentration a second specific binding to (G-C) pairs most probably through hydrogen bonding is established. Viscometric results suggest a distamycin-induced local bending of the helix and could support the idea of a preferential alignment of the ligand molecule along only one strand in the groove which differs from the netropsin interaction mechanism. The possibility of an overlapping binding of the oligopeptides in the small groove is discussed.     

一个新的粗糙脉孢霉基因组DNA制备方法

粗糙脉孢霉基因组DNA的制备多数费工费时,并且需要液氮研磨。而一般实验室没有液氮设备。本文提供了一个不需要液氮研磨就可以快速制备粗糙脉孢霉基因组DNA的方法,使用裂解液、石英砂和苯酚振荡裂解细胞壁,然后用苯酚／氯仿抽提DNA。利用PCR从基因组扩增出粗糙脉孢霉Ⅰ号染色体右臂上的校孔转运蛋白基因片段。     

Isolation and genetic analysis of nuclease halo (nuh) mutants of Neurospora crassa.

Summary Nuclease halo (nuh) mutants of the ascomycete Neurospora crassa have been isolated which are characterized reduced release of deoxyribonuclease (DNase) activities from colonies grown on sorbose-containing agar media. To identify nuh mutants, mutagenized isolates were transferred to commercial DNase test agar, or grown on minimal medium and then overlayed with agar that contained heat-denatured DNA. DNase activity was visualized by acid precipitation which produced clear rings of digestion (haloes) around the colonies.To identify the number of genes in which mutations lead to reduced release of nuclease activity, eleven nuh mutants were checked for close linkage and linked pairs were tested for complementation. These mutants were assigned to eight genes, and all except one were mapped in six small regions of the Neurospora linkage maps. In addition, among a large number of existing mutants which were tested for nuclease haloes, two mutants were found that showed the Nuh phenotype, namely uvs-3 and uvs-6. One of the isolated nuh mutants was also found to be sensitive to UV and was mapped close to uvs-3; it may represent a new allele of this gene.As a first step towards identification of genuine nuclease mutants, extensively backcrossed strains of mutants from different genes have been assayed for nuclease activity with denatured DNA in extracts. A pronounced reduction, compared to wild type at the same stage of growth, was found in uvs-3 and also in nuh-3, a mutant that is not UV-sensitive.     

Conformational transitions and hydration of poly d (A-T) · poly d (A-T) in fibers

Conformational transitions of poly d(A-T) · poly d (A-T) have been studied by fiber X-ray diffraction and measurement of fiber dimensions. Results obtained for the D-A-B and D-B transitions are presented and analyzed. For all these form transitions, cooperativity effects are observed for the variation of the rise per nucleotide versus the relative humidity. Detailed information about hydration of the polynucleotide during form transitions and the numbers of water molecules per nucleotide necessary to stabilize the different helical conformations are presented. Offprint requests to: S. Premilat     

A poly d(A-T)-unwinding glycoprotein from roe-deer liver.

A DNA double-helix destabilizing protein was purified from roedeer liver to near homogeneity. It is a glycoprotein containing fucose, xylose, mannose and at least two undefined shugars. For comparative work an unwinding coefficient was defined and determined for different doublestranded polynucleotides. The present protein revealed a remarkable preference for unwinding A-T deoxybase pairs.     

Helix coil transitions of d (A)n·d(T)n,d(A-T)n·d(A-T)n,and d(A-A-T)n·d(A-T-T)n; evaluation of parameters governing DNA stability

The thermally induced helix-coil transitions of three A-T DNAs, d(A)_n·d(T)_n, d(A-T)_n·d(A-T)_n, and d(A-A-T)_n·d(A-T-T)_n, were studied. Experimental transition curves of the DNAs were analyzed using the loop entropy model of DNA melting. The calculation of the melting curve of d(A-A-T)_n·d(A-T-T)_n is presented using the integral equation formalism of Goel and Montroll. The aim of this work was to evaluate thermodynamic parameters which govern DNA stability and to test the theoretical model employed in the analysis. Our results show (1) an excellent over-all agreement between theory and experiment, (2) a loop entropy exponent k = 1.55 ± 0.05 provided the best fit to all the polymer transition curves, (3) the evaluated stacking free energies reflect the relative stability of the DNAs, and (4) the stacking energies of the ApA·TpT dimer evaluated from d(A)_n·d(T)_n and d(A-A-T)_n·d(A-T-T)_n differ. The last result is consistent with different conformations for the dimer in these two polymers.     

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.     

Synthesis of signals for de novo DNA methylation in Neurospora crassa

Most 5-methylcytosine in Neurospora crassa occurs in A:T-rich sequences high in TpA dinucleotides, hallmarks of repeat-induced point mutation. To investigate how such sequences induce methylation, we developed a sensitive in vivo system. Tests of various 25- to 100-bp synthetic DNA sequences revealed that both T and A residues were required on a given strand to induce appreciable methylation. Segments composed of (TAAA)(n) or (TTAA)(n) were the most potent signals; 25-mers induced robust methylation at the special test site, and a 75-mer induced methylation elsewhere. G:C base pairs inhibited methylation, and cytosines 5' of ApT dinucleotides were particularly inhibitory. Weak signals could be strengthened by extending their lengths. A:T tracts as short as two were found to cooperate to induce methylation. Distamycin, which, like the AT-hook DNA binding motif found in proteins such as mammalian HMG-I, binds to the minor groove of A:T-rich sequences, suppressed DNA methylation and gene silencing. We also found a correlation between the strength of methylation signals and their binding to an AT-hook protein (HMG-I) and to activities in a Neurospora extract. We propose that de novo DNA methylation in Neurospora cells is triggered by cooperative recognition of the minor groove of multiple short A:T tracts. Similarities between sequences subjected to repeat-induced point mutation in Neurospora crassa and A:T-rich repeated sequences in heterochromatin in other organisms suggest that related mechanisms control silent chromatin in fungi, plants, and animals.