Structure of the human hemopexin gene and evidence for intron-mediated evolution

Summary The human hemopexin gene was isolated and its structure determined. The gene spans approximately 12 kb and is interrupted by nine introns. When the intron/exon pattern was examined with respect to the polypeptide segments they encode, a direct correspondence between exons and the 10 repeating units in the protein was observed. The introns are not randomly placed; they fall in the middle of the region of amino acid sequence homology in strikingly similar locations in 6 of the 10 units and in a symmetrical position in the two halves of the coding sequence. These features strongly support the hypothesis that the gene evolved through intron-mediated duplications of a primordial sequence to a five-exon cluster. A more recent gene duplication led to the present-day gene organization.     

The hemopexin gene maps to the same location as the beta-globin gene cluster on human chromosome 11

Using human hemopexin cDNA clones isolated from lambda gt11 cDNA library as probes, we have carried out Southern blot analysis of a series of human-Chinese hamster somatic cell hybrids containing different combinations of human chromosomes. Synteny analysis revealed 100% concordance between the hemopexin gene and human chromosome 11. In situ hybridization of 3H-labeled hemopexin cDNA to metaphase chromosomes prepared from human lymphocytes further localized the gene to the region p15.4-p15.5, the same location as the beta-globin gene cluster.     

Differential expression pattern of coq-8 gene during development in Caenorhabditis elegans

Coenzyme Q (Q) and the genes involved in its biosynthesis are involved in aging and development of Caenorhabditis elegans. Q is synthesized by at least eight highly conserved nuclear coq genes, but this biosynthesis pathway and its regulation is not known. The coq-8 gene sequence has homology to the ABC-1 family kinases and is the only known candidate for a possible regulation of this pathway. To study coq-8 expression pattern, we have developed a C. elegans transgenic strain expressing ubiquinone biosynthesis coq-8 gene promoter and GFP construct. We show here an age-dependent specific pattern from embryo to senescence for COQ-8 protein expression. Expression in embryo was triggered by a defined group of blastomers before morphogenesis. In elderly nematodes expression was only observed in nervous system, whilst expression in larvae was also detected in hypodermis, muscles and coelomocytes. Global expression provide a regulated pattern during life cycle of the nematode.     

Characterization of sheep hemopexin glycovariants

The hemopexin phenotype HpxB1 isolated from sheep serum, yields three major bands when subjected to starch gel and/or polyacrylamide gel electrophoresis which are here designated as subcomponents HpxB1-I, HpxB1-II and HpxB1-III. Electrospray mass spectrometric analysis of samples of the isolated subcomponents prepared by ion exchange chromatography showed that each was composed of three glycoproteins and that the major difference between the subcomponents was due to their constituent glycoproteins possessing different numbers of sialic acid residues. Combined analysis of the ESI-MS data and of the overall carbohydrate compositional data obtained by colorimetric procedures, leads to the composition of the glycan of each glycoprotein, and a combined methylation and 400 MHz H-NMR analysis of the alkaline cleaved glycans identified them as being of only the biantennaryN-acetyllactosamine type. Taking into account the molecular mass, the carbohydrate content and structure it may be concluded that each of the constituent glycoproteins contain fiveN-glycosidically linked glycans.Abbreviations HpxB1 hemopexin phenotype B1 - Man mannose - Gal galactose - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - GlcNAc-ol N-acetylglucosaminitol     

辣椒疫霉RXLR型效应子PcAvh2的序列多态性、基因转录特征及功能

【目的】分析辣椒疫霉中RXLR型效应子PcAvh2的序列多态性,研究该效应子在辣椒疫霉生长发育和侵染阶段的转录特征及其生物学功能。【方法】本研究通过高保真扩增,分析2个烟草疫霉、1个恶疫霉和31个辣椒疫霉菌株的PcAvh2序列;提取辣椒疫霉菌丝、游动孢子囊、游动孢子、萌发休止孢和7个侵染时间点(1.5、3、6、12、24、36、72 h)的本氏烟根部总RNA,利用RT-qPCR分析PcAvh2的转录表达水平;利用PVX瞬时表达系统,分析PcAvh2是否抑制6种效应子(BAX、INF1、PsojNIP、PsCRN63、PsAvh241、R3a/Avr3a)激发的植物免疫反应;利用CaCl_2-PEG介导的原生质体稳定转化技术,沉默PcAvh2基因,分析辣椒疫霉致病力的变化。【结果】PcAvh2为典型的RXLR效应子,在辣椒疫霉群体中该效应子具有10个等位基因,而且烟草疫霉和恶疫霉中也存在该效应子。该基因在辣椒疫霉的侵染阶段上调表达,它能够抑制6种效应子激发的植物免疫反应,进一步研究发现基因沉默导致辣椒疫霉的致病力显著下降。【结论】RXLR型效应子PcAvh2是辣椒疫霉中一个重要的侵染致病因子。     

Use of hemopexin domains and monoclonal antibodies to hemopexin to probe the molecular determinants of hemopexin-mediated heme transport

Plasmin cleaves rabbit serum apohemopexin (Mr = 60,000) at a single site producing a heme-binding domain (I, Mr = 35,000) and a second domain (II, Mr = 25,000) (W. T. Morgan and A. Smith (1984) J. Biol. Chem. 259, 12001-12005). The absorbance spectra of heme-domain I are indicative of a bis-histidyl coordination complex with the central heme iron atom. Chemical modification of the 5 histidine residues of apo-domain I with diethylpyrocarbonate abolished heme binding, supporting this assignment. Upon binding heme, domain I migrates more rapidly in sucrose gradients, and, in sedimentation velocity experiments, the s value of domain I increases from 3.17 +/- 0.04 to 3.71 +/- 0.09, a notably large increase which indicates that the domain becomes much more compact. This conformational change which plays a pivotal role in hemopexin function requires the bis-histidyl coordination with heme iron and leads to a tighter association between domain I and domain II shown by the co-migration of heme-domain I and domain II in sucrose gradients. In turn, the association of heme-domain I with domain II increases the thermal stability of the heme-domain I chromophore. Results of binding studies using mouse hepatoma cells and isolated domains indicate that domain I not only binds heme but also plays a vital part in the hemopexin-receptor interaction. The change in conformation of domain I upon heme binding and the association between domains I and II induced by heme are both notable determinants of the strength of the hemopexin-receptor interaction, but an intact "hinge region" between the domains is not necessary for receptor binding. The importance of both domains in bringing about the transport function of hemopexin is confirmed by the ability of three (two specific for domain I and one for domain II) of seven monoclonal antibodies raised against hemopexin to inhibit the hemopexin-receptor interaction.