Probing the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) by mutational analysis

Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from Pseudomonas fluorescens Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.     

Genetic analysis of fertility-restorer genes in rice

Wild-abortive (WA), Honglian (HL) and Baro-II (BT) are three important cytoplasmic male sterility (CMS) types in rice. It is essential to investigate genetic mode and allelism of fertility restorer (Rf) genes and the relationship between Rf and CMS. Fertility of the all test-cross F₁ plants shows that the restorer-maintainer relationship is similar for HL-CMS and BT-CMS, while that is variance for WA-CMS and HL-CMS (or BT-CMS), respectively. Genetic analysis of Rf genes indicates that HL-or BT-CMS are controlled by single dominant Rf gene and WA-CMS is controlled by one or two pairs of dominant Rf genes, which reflects the characters of the gametophytic and sporophytic restoration CMS type. It is concluded that there are at least three Rf loci in different accessions with Rf genes for each CMS type.     

Organization of phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides.

A heterologous phosphoribulokinase (PRK) gene probe was used to analyze two recombinant plasmids isolated from a Rhodopseudomonas (Rhodobacter) sphaeroides gene library. These plasmids were previously shown to carry the genes for form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPC/O). Southern blot hybridization analysis indicated that there were two PRK genes linked to the RuBPC/O coding sequences. Restriction mapping showed the arrangement of the duplicate sets of PRK and RuBPC/O to be distinct. Subcloning of the hybridizing PRK sequences downstream of the lac promoter of pUC8 allowed expression of the two PRK enzymes in Escherichia coli. Analysis of the purified proteins by sodium dodecyl sulfate-slab gel electrophoresis revealed polypeptides with molecular weights of 32,000 and 34,000 corresponding to the form I and form II PRKs, respectively. Preliminary experiments on sensitivity to NADH regulation suggested that the two PRK enzymes differ in catalytic properties.     

Increased glycopeptide production after overexpression of shikimate pathway genes being part of the balhimycin biosynthetic gene cluster

Amycolatopsis balhimycina produces the vancomycin-analogue balhimycin. The strain therefore serves as a model strain for glycopeptide antibiotic production. Previous characterisation of the balhimycin biosynthetic cluster had shown that the border sequences contained both, a putative 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (dahp), and a prephenate dehydrogenase (pdh) gene. In a metabolic engineering approach for increasing the precursor supply for balhimycin production, the dahp and pdh genes from the biosynthetic cluster were overexpressed both individually and together and the resulting strains were subjected to quantitative physiological characterisation. The constructed strains expressing an additional copy of the dahp gene and the strain carrying an extra copy of both dahp and pdh showed improved specific glycopeptide productivities by approximately a factor three, whereas the pdh overexpression strain showed a production profile similar to the wild type strain. In addition to the overexpression strains, corresponding deletion mutants, Δdahp and Δpdh, were constructed and characterised. Deletion of dahp resulted in significant reduction in balhimycin production whereas the Δpdh strain had production levels similar to the parent strain. Based on these results the relation between primary and secondary metabolism with regards to Dahp and Pdh is discussed.     

Genetic analysis of non-essential bacteriophage T7 genes

Isolation and genetic characterization of a series of deletions and point mutants affecting two non-essential genes of bacteriophage T7 is described. The T7 ligase gene falls between genes 1 and 2, and is designated gene 1.3. Another non-essential gene, designated gene 0.7, has been mapped to the left of gene 1. In order to facilitate isolation and characterization of these mutants, host strains were found in which one or both of these T7 genes is required for growth.     

水稻窄叶突变体zy17的遗传分析和候选基因鉴定

器官大小调控是一个基本的发育生物学过程,受细胞分裂和细胞扩展的影响。然而,植物器官大小调控的遗传和分子机理仍不清楚。为了进一步了解器官大小调控的分子机制,文章分离了一系列水稻叶子宽窄改变的突变体。其中,窄叶突变体zy17叶变窄,同时伴有植株矮化、穗子变小、枝梗数和穗粒数降低的表型。遗传分析表明该窄叶性状受1个隐性基因控制;细胞学分析表明该突变体叶子的细胞数目和维管束数目显著降低,表明ZY17影响了细胞分裂。基因组重测序进一步筛选出ZY17的3个候选基因：Os02g22390基因突变发生在内含子区,编码蛋白为逆转座蛋白;Os02g28280和Os02g29530基因突变都发生在外显子区,其中Os02g28280编码一个功能未知蛋白,该基因突变后,发生碱基置换,产生非同义突变;Os02g29530编码一个含糖基转移酶相关的PFAM结构域的蛋白,该基因突变后,出现两个碱基的缺失,从而导致其蛋白翻译提前终止。对候选基因的深入研究,将揭示水稻叶子大小调控的机制。     

Genetic analysis of the Drosophila Gs(alpha) gene.

One of the best understood signal transduction pathways activated by receptors containing seven transmembrane domains involves activation of heterotrimeric G-protein complexes containing Gs(alpha), the subsequent stimulation of adenylyl cyclase, production of cAMP, activation of protein kinase A (PKA), and the phosphorylation of substrates that control a wide variety of cellular responses. Here, we report the identification of "loss-of-function" mutations in the Drosophila Gs(alpha) gene (dgs). Seven mutants have been identified that are either complemented by transgenes representing the wild-type dgs gene or contain nucleotide sequence changes resulting in the production of altered Gs(alpha) protein. Examination of mutant alleles representing loss-of-Gs(alpha) function indicates that the phenotypes generated do not mimic those created by mutational elimination of PKA. These results are consistent with the conclusion reached in previous studies that activation of PKA, at least in these developmental contexts, does not depend on receptor-mediated increases in intracellular cAMP, in contrast to the predictions of models developed primarily on the basis of studies in cultured cells.     

Genetic mapping of gray leaf spot (GLS) resistance genes in maize

Bulked segregant analysis was used to identify amplified fragment length polymorphism markers (AFLPs) linked to quantitative trait loci (QTLs) involved in the resistance to gray leaf spot (GLS) in maize. By using ten AFLP primer combinations 11 polymorphic markers were identified and converted to sequence- specific PCR markers. Five of the 11 converted AFLPs were linked to three GLS resistance QTLs. The markers were mapped to maize chromosomes 1, 3 and 5 using existing linkage maps of two commercially available recombinant inbred-line populations. Converted restriction fragment length polymorphism markers and microsatellite markers were used to obtain a more-precise localization for the detected QTLs. The QTL on chromosome 1 was localized in bin 1.05/06 and had a LOD score of 21. A variance of 37% was explained by the QTL. Two peaks were visible on chromosome 5, one was localized in bin 5.03/04 and the other in bin 5.05/06. Both peaks had a LOD score of 5, and 11% of the variance was explained by the QTLs. A variance of 8–10% was explained by the QTL on chromosome 3 (bin 3.04). The consistency of the QTLs was tested across two F₂ populations planted in consecutive years. Received: 10.10.00 / Accepted: 26.01.01     

核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)

文章就核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的分布、结构、性质、分类与功能的研究进展作了介绍。     

水稻斑点叶突变体spl101和spl102的筛选及候选基因鉴定

斑点叶突变体是指植物在正常生长条件下叶片或叶鞘上自发形成、且与病原菌侵染产生的病斑类似的一类突变体,筛选并研究斑点叶突变体对揭示植物抗病反应机理具有重要意义。为了进一步研究斑点叶的形成机制,本文通过EMS诱变品种宽叶粳(KYJ),筛选得到两个斑点叶突变体spl101和spl102。这两个突变体在生长发育晚期(抽穗期以后)形成严重的类病斑。遗传分析表明,spl101和spl102均受隐性单基因控制。利用Mutmap方法对候选基因进行克隆,结果显示,spl101和spl102的候选基因均为OsEDR1,该基因与类病斑发生有关。在spl101中,OsEDR1基因突变发生在第6外显子和第6内含子的交接处,该突变导致第6内含子的错误识别,最终造成移码突变。在spl102中,OsEDR1基因突变发生在第10外显子上,导致一个苯丙氨酸(F)变成半胖氨酸(C)。因此,本研究鉴定了两个新的OsEDR1等位突变,对OsEDR1抗病反应机理的进一步研究及丰富水稻种质资源具有积极意义。同时验证了利用Mutmap方法克隆水稻突变基因的有效性。     

Further biochemical studies on aminopyrrolnitrin oxygenase (PrnD)

Active site modeling of dimerization interface in combination with site-directed mutagenesis indicates that the electron in the PrnD Rieske oxygenase can be transferred by either of two pathways, one involving Asp183′ and the other involving Asn180′. In addition, the overexpression of the isc operon involved in the assembly of iron-sulfur clusters increased the catalytic activity of PrnD in Escherichia coli by a factor of at least 4.     

Genetic fine localization of the β-glucocerebrosidase (GBA) and prosaposin (PSAP) genes: implications for Gaucher disease

Mutations in the glucocerebrosidase (GBA) and prosaposin (PSAP) genes are responsible for Gaucher disease, the most prevalent sphingolipidosis. Somatic cell hybrid analysis and in situ hybridization experiments have localized the GBA gene to 1q21 and the PSAP gene to 10q21-q22. We performed pairwise and multi-point linkage analyses between the two genes and several highly polymorphic markers from the Généthon human linkage map. Our results show that six markers cosegregate with the GBA gene (Z_max = 8.73 at θ = 0.00 for marker D1S2714) and define a 3.2-cM interval between D1S305 and D1S2624 as the most probable location for the gene. Three of these markers (D1S2777, D1S303, and D1S2140), as well as the gene encoding pyruvate kinase (PKLR), are contained in a single YAC clone together with the GBA gene. A new polymorphism was identified within the PSAP gene (C16045T) and used for linkage studies. The multi-point analysis places the gene in a 9.8-cM interval between D10S1688 and D10S607. The fine localization of these genes provides a useful tool for cosegregation analysis, indirect molecular diagnosis, and population genetic studies. Received: 22 October 1996 / Accepted: 4 February 1997