Interactions between subdomains in the partially folded state of staphylococcal nuclease

Staphylococcal nuclease can be roughly divided into a beta-subdomain in N-terminal and an alpha-subdomain in C-terminal. They fold sequentially under certain conditions, causing a partially folded intermediate state in which the native-like beta-barrel persists while alpha-helix regions largely disorder. To investigate the possible long-range interactions between the two subdomains in the intermediate, N-terminal fragments have been used as intermediate analogues, with polypeptide ending at positions 102, 110, 121 and 135 and with a tryptophan substitution at position 66 or 88 to facilitate the observation of the beta-barrel. Segment-resolved interactions between beta-barrel and residues 103-135 were identified by comparing their spectroscopic properties of fluorescence, circular dichroism and NMR and by their stability. Except for unstable V66W102, the guanidine and thermal denaturation of fragments are cooperative and well approximated by the two-state transition. Minimal stable structure units of both tryptophan-containing fragments comprise residues 1-110. With the main interaction in segment 103-135, residues 103-110 contribute approximate 2 kcal/mol to the stability. Elongation of C-terminal from 110 residue neither increases the stability nor alters the structure core of the G88W fragments. However, residues 111-121 influence the tertiary structure of the V66W fragments suggesting its minor interactions with beta-barrel.     

Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants

The biosynthesis of lignin in vascular plants is regulated both developmentally and environmentally. In the inflorescence stems of Arabidopsis, lignin is mainly deposited in the walls of xylem cells and interfascicular fiber cells during normal plant growth and development. The mechanisms controlling the spatial deposition of lignin remain unknown. By screening ethyl methanesulfonate-mutagenized populations of Arabidopsis, we have isolated two allelic elp1 (ectopic deposition of lignin in pith) mutants with altered lignin deposition patterns. In elp1 stems, lignin was ectopically deposited in the walls of pith parenchyma cells in addition to its normal deposition in the walls of xylem and fiber cells. Lignin appeared to be deposited in patches of parenchyma cells in the pith of both young and mature elp1 stems. The ectopic deposition of lignin in the pith of elp1 stems was accompanied by an increase in the activities of enzymes in the lignin biosynthetic pathway and with the ectopic expression of caffeoyl coenzyme A O-methyltransferase in pith cells. These results indicate that the ELP1 locus is involved in the repression of the lignin biosynthetic pathway in the pith. Isolation of the elp1 mutants provides a novel means with which to study the molecular mechanisms underlying the spatial control of lignification.     

cDNA cloning by amplification of circularized first strand cDNAs reveals non-IRE-regulated iron-responsive mRNAs

Currently, the rapid amplification of cDNA ends (RACE) is the most common method for PCR cloning of cDNA. Because RACE uses a gene specific primer and one adaptor primer that is shared by all cDNAs may result in numerous nonspecific products that can hinder the cloning process. Here we report a new method that uses circularized first strand cDNA from mRNA and two gene specific primers to amplify both the 5' and 3' cDNA ends in one reaction. A cDNA band of correct size can be obtained on the first pass in this approach. If the correct size is not obtained on the first pass, amplification of cDNA ends can be repeated until the correct size of the cDNA is obtained. We tested this new method on eight mRNAs that we have previously shown to respond to cellular iron levels. We obtained sequences for six mRNAs that were 43 bp to 1324 bp longer than that reported in GenBank and obtained the same length sequence for the other two mRNAs. RNA folding program shows no iron responsive elements (IRE) on these mRNA. In conclusion, our cloning approach offers a more efficient method for cloning full-length cDNA and it may be used to replace the existing method of 5' end cDNA extension. The data enabled us to exclude the possibility that the expression of these iron responsive genes are regulated by IREs.     

Sativin: a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon

An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. The procedure entailed extraction, affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It possessed an N-terminal amino acid sequence which showed similarity to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from Pisum sativum var arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50= 14 microM). Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus and Pleurotus ostreatus but not against Rhizoctonia solani.     

73例中国人血友病甲基因突变的分析

我们用Southern blotting、PCR、变性梯度凝胶电泳(DGGE)和DNA测序等方法对73例血友病甲患者(经上海瑞金医院测定血浆FVⅢ:C和vWF:Ag诊断,其中无亲缘关系患者65例,按FVⅢ:C水平分为轻、中、重三型。FVⅢ:C< 2%为重型,共47例;FVⅢ:C 2%-5%为中型,共9例;FVⅢ:C5%-25%为轻型,共1 7例)进行FVⅢ基因突变检测。共检出内含子22倒位23例,均为重型,约占重型的49%,与国外报道相似。余下50例(其中无亲缘关系者45)用PCR-DGG E分析所有外显子及其侧翼内含子序列,发现异常条带则进行DNA测序。在17例患者中检出突变13种,其中无义突变5种,均为重型;错义突变6种,除1例外都是轻中型;小缺失2例,都是重型;其中,AA466Lys(AAG)-Thr(ACG)、719Tyr(TAC)-Stop(TAG)、AA826 Asp(GAC)-Glu(GAA)、312Ile(ATC)-xxC及AA1551-1552del(AGAA)为新发现的突变。有亲缘关系的患者都有相同的基因突变,而在无亲缘关系患者未发现相同突变。基因突变与临床表现基本相符。 Abstract:We use Southern blotting,PCR,denaturing gradient gel electrophoresis(DGGE)and DNA sequencing to detect gene mutations of haemophilia A in Chinese population.73 cases(47 severe)(FVIII:C<2%),9 moderate(FVIII:C 2%~5%),17 mild(FVIII:C 5%~25%)of haemophilia A were first screened with Southern blotting,23 were found to be the intron 22 inversion type,all being severe cases.The remaining 50 cases without intron 22 in version were examined with PCR-DGGE.Genomic DNA were amplified using GC-clamped primers covering all the exons and all flanking intron regions.Abnormal bands were sequenced.13 different mutations were identified,including 5 nonsense mutations,6 missense mutations and 2 small deletions.5 mutations,AA466Lys(AAG)-Thr(ACG),AA719Tyr(TAC)-Stop(TAG),AA826Asp(GAC)-Glu(GAA),AA312Ile(ATC)-xxC and AA1551-1552del(AGAA)have not been reported before.Generally the genetic defects correspond to the clinical conditions.     

陆地棉枯萎病抗性基因的等位性测定及连锁分析

1995-1996年,对我国育成的有代表性的5个抗病品种进行抗枯萎病基因的等位性测定。结果表明:在所选用的5个抗病品种中至少存在两个不同的抗病基因(暂定名为Fwl和Fw2)。连锁分析显示:Fwl与T586的8个标志性状间、Fw2与T582、T586的13个标志性状间无连锁关系。 Abstract:Allelism in vestigation of genes resistante to Fusarium wilt in cotton suggested that there were 2 genes(assigned symbols Fw1 and Fw2)in 5 cultivars used.No linkage was found between Fw1 and the marker genes in T586 and between Fw2 and those marker genes in T582 and T586.