The effect of Ph gene alleles on synaptonemal complex formation in Triticum aestivum × T. kotschyi hybrids

Summary Chromosome pairing at zygotene-pachytene was studied in Triticum aestivum × T. kotschyi hybrids (2n=5x=35, genomic constitution ABDC^US^v) by electron microscopy of synaptonemal complexes in spread microsporocyte nuclei. Hybrids carrying either the Ph allele or the ph allele, which differ markedly in metaphase I pairing, are both capable of greater than 90% pachytene pairing, although pairing in the Ph hybrids appeared slower or less synchronised. In both genotypes branched synaptonemal complexes were formed by intra-and interchromosomal pairing. The Ph gene control on homoeologous pairing does not act on the ability to pair into synaptonemal complexes. It may act on the rate of pairing or the time of crossing over.     

Internode length in Pisum. Gene lkd

A new, single gene, recessive internode length mutant in Pisum, lkd, is described. The internodes of lkd plants are ca. 40% shorter than comparable Lkd plants and this difference appears greater in the dark than in the light. The mutant does not appear to be dwarfed due to modified gibberellin (GA) levels, as determined by gas-chromatography-selected ion monitoring (GC-SIM) for GA₁ and GA₂₀. In relative terms, the mutant responds as well as the wild-type to applied GA₁. However due to its initial short stature it does not elongate to the same extent as the wild-type to high doses of GA₁ suggesting that some other factor, unrelated to GA levels or perception is probably limiting growth in this mutant. Author for correspondence     

Paternal H3K4 methylation is required for minor zygotic gene activation and early mouse embryonic development

Epigenetic modifications, such as DNA methylation and histone modifications, are dynamically altered predominantly in paternal pronuclei soon after fertilization. To identify which histone modifications are required for early embryonic development, we utilized histone K‐M mutants, which prevent endogenous histone methylation at the mutated site. We prepared four single K‐M mutants for histone H3.3, K4M, K9M, K27M, and K36M, and demonstrate that overexpression of H3.3 K4M in embryos before fertilization results in developmental arrest, whereas overexpression after fertilization does not affect the development. Furthermore, loss of H3K4 methylation decreases the level of minor zygotic gene activation (ZGA) predominantly in the paternal pronucleus, and we obtained similar results from knockdown of the H3K4 methyltransferase Mll3/4. We therefore conclude that H3K4 methylation, likely established by Mll3/4 at the early pronuclear stage, is essential for the onset of minor ZGA in the paternal pronucleus, which is necessary for subsequent preimplantation development in mice.     

Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

Background

Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.

Results

We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.

Conclusions

This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users.     

弗氏柠檬酸菌1,3-丙二醇合成的代谢工程研究

【目的】研究弗氏柠檬酸菌(Citrobacter freundii) 1,3-丙二醇合成的代谢过程。【方法】构建甘油脱氢酶基因GSR-lacZ、1,3-丙二醇氧化还原酶基因PDO-lacZ和甘油脱水酶基因GL-lacZ等报告基因。在此基础上,构建3个相应的转座子突变文库。【结果】筛选到6株突变子,其相应关键酶表达水平提高1?11倍,1,3-丙二醇产量提高幅度为3%?50%。对转座子插入位点分析显示,5株突变子插入位点均为β-内酰胺酶(CKO_02592)编码基因,1株突变子插入位点为二氢硫辛酰胺基转移酶(CKO_02433)编码基因。进一步分析发现,β-内酰胺酶基因突变显著提高甘油脱水酶和甘油脱氢酶的表达水平,而1,3-丙二醇氧化还原酶表达水平没有变化;二氢硫辛酰胺基转移酶基因突变显著提高1,3-丙二醇氧化还原酶表达水平,其他两种关键酶基因表达水平不变。【结论】β-内酰胺酶和二氢硫辛酰胺基转移酶基因能够分别影响1,3-丙二醇合成代谢途径关键酶的表达,为构建工程菌株打下基础。     

The deubiquitinating enzyme AMSH1 is required for rhizobial infection and nodule organogenesis in Lotus japonicus

Legume–rhizobium symbiosis contributes large quantities of fixed nitrogen to both agricultural and natural ecosystems. This global impact and the selective interaction between rhizobia and legumes culminating in development of functional root nodules have prompted detailed studies of the underlying mechanisms. We performed a screen for aberrant nodulation phenotypes using the Lotus japonicus LORE1 insertion mutant collection. Here, we describe the identification of amsh1 mutants that only develop small nodule primordia and display stunted shoot growth, and show that the aberrant nodulation phenotype caused by LORE1 insertions in the Amsh1 gene may be separated from the shoot phenotype. In amsh1 mutants, rhizobia initially became entrapped in infection threads with thickened cells walls. Some rhizobia were released into plant cells much later than observed for the wild‐type; however, no typical symbiosome structures were formed. Furthermore, cytokinin treatment only very weakly induced nodule organogenesis in amsh1 mutants, suggesting that AMSH1 function is required downstream of cytokinin signaling. Biochemical analysis showed that AMSH1 is an active deubiquitinating enzyme, and that AMSH1 specifically cleaves K63‐linked ubiquitin chains. Post‐translational ubiquitination and deubiquitination processes involving the AMSH1 deubiquitinating enzyme are thus involved in both infection and organogenesis in Lotus japonicus.     

拟南芥高亲和性K~+转运蛋白AtHAK5功能位点的鉴定

AtHAK5是拟南芥高亲和性钾转运体,其基因表达受低钾条件强烈诱导,编码蛋白在低钾条件下可以整合到质膜.生物信息学分析发现其氨基酸序列含有多处潜在的磷酸化位点.本研究假设这些位点对于AtHAK5的功能至关重要,为探讨AtHAK5的功能位点,分别将AtHAK5 cDNA和带有13种不同点突变位点的AtHAK5转化到athak5突变体中,获得14种稳定表达的转基因植株.利用athak5突变体根对Cs敏感的表型,最终确定T549A和T666A为非核心磷酸化位点.如下11个位点为AtHAK5功能必需位点:F85L,T86A,T311A,T359A,P551S,D552N,S603A,S604A,K668E,S698A和V713L.     

Antibodies and protein misfolding: From structural research tools to therapeutic strategies

Protein misfolding disorders, including the neurodegenerative conditions Alzheimer's disease (AD) and Parkinson's disease (PD) represent one of the major medical challenges or our time. The underlying molecular mechanisms that govern protein misfolding and its links with disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species within the crowded environment of the cell and tissue. Nevertheless, much progress has been made in understanding these events in recent years through innovative experiments and therapeutic strategies, and in this review we present an overview of the key roles of antibodies and antibody fragments in these endeavors. We discuss in particular how these species are being used in combination with a variety of powerful biochemical and biophysical methodologies, including a range of spectroscopic and microscopic techniques applied not just in vitro but also in situ and in vivo, both to gain a better understanding of the mechanistic nature of protein misfolding and aggregation and also to design novel therapeutic strategies to combat the family of diseases with which they are associated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

小麦矮秆圆粒突变体的鉴定与分析

小麦的子粒形态性状和株高与小麦的产量密切相关,是育种的重要选择目标性状。本研究通过对小麦品种偃展1号(YZ1)EMS突变体W98的农艺性状的调查与分析,发现W98的株高只有24 cm,而野生型YZ1的株高是73 cm。突变体株高的变异是由每个节间长度变短造成的,而非节间数目减少所致。相关分析表明矮秆与圆粒性状呈显著相关。利用高秆长粒的墨西哥品种10th12与突变体W98配制杂交组合,获得1544个F2∶3单株(株系),通过对分离群体的遗传分析,发现圆粒性状是由1对不完全显性基因控制的。赤霉素(GA)与油菜素内酯(BR)激素敏感性试验表明:野生型和突变体都对GA处理不敏感;不同浓度BR的展叶试验表明野生型对BR不敏感,而突变体W98对BR敏感。