Distinct regions of the chicken p46 polypeptide are required for its in vitro interaction with histones H2B and H4 and histone acetyltransferase-1

We cloned cDNA encoding the chicken p46 polypeptide, chp46, homologous to the p48 subunit of chicken chromatin assembly factor-1, chCAF-1p48. It comprises 424 amino acids including a putative initiation Met, is a member of the WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to chCAF-1p48 and 94.3% identity to the human and mouse p46 polypeptides (hup46 and mop46). The in vitro immunoprecipitation experiment established that chp46 interacts with histones H2B and H4 and chicken histone acetyltransferase-1, chHAT-1, whereas hup46 interacts with histones H2A and H4 and chHAT-1 and chCAF-1p48 with histone H4 and chHAT-1. The in vitro immunoprecipitation experiment, involving truncated mutants of chp46, revealed not only that two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. Furthermore, the GST pulldown affinity assay, involving truncated mutants of chp46, revealed that a region comprising amino acids 359-404 (in fact, 375-404) binds to chHAT-1 in vitro. Taken together, these results indicate not only that chp46 should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with chHAT-1 and histones H2B and H4, but also that the proper propeller structure of chp46 is not necessary for its interaction with chHAT-1.     

日本血吸虫中国大陆株抱雌沟蛋白编码基因的克隆和表达

根据曼氏血吸虫抱雌沟蛋白SmGcp序列和日本血吸虫编码抱雌沟蛋白保守区的基因片段jGcp1分别设计三对引物,以日本血吸虫中国大陆株成虫mRNA为模板 ,用RT－PCR法扩增出大小为1949bp的基因片段。经序列分析推断该基因片段含编码日本血吸虫抱雌沟蛋白基因的阅读框,与SmGcp碱基一致性为85％,其理论推测氨基酸组成与曼氏血吸虫抱雌沟蛋白的一致性为83．7％。将上述扩增的基因片段克隆到表达载体pET28c( )中,在大肠杆菌BL21中获得表达,融合表达产物分子量约为80kD。利用日本血吸虫成虫抗原免疫血清对该表达产物进行Western印迹检测,在预测位置出现了明显的识别条带,说明该编码日本血吸虫中国大陆株抱雌沟蛋白基因的表达产物具有抗原性。     

球毛壳菌组蛋白H3基因克隆与特性分析

构建了球毛壳菌菌丝的cDNA文库,并获得了1410条ESTs序列,用里氏木霉(Hypocrea jecorina,AAM76068)和粗糙脉胞菌(Neurospora crassa,CAA25761)的组蛋白H3基因(Histone H3)蛋白序列对球毛壳菌(Chaetomium globosum)ESTs序列本地数据库进行tBlastn检索,获得了球毛壳菌组蛋白H3cDNA序列。cDNA序列全长739bp,开放阅读框411bp,编码136个氨基酸组成的多肽,蛋白分子量为15.4kD。BlastP同源性分析表明该基因与里氏木霉同源性最高为100%;与地钱(Marchantia polymorpha)同源性最低为95%。三级结构预测表明,该蛋白C端为球状结构域,而N端结构对其发挥调控作用起重要作用。该基因的cDNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY669068,AAT74576)。     

Schistosoma mansoni tropomyosin: cDNA characterization, sequence, expression, and gene product localization

We have defined the polypeptide pattern of 3-hr Schistosoma mansoni schistosomula on nonequilibrium two-dimensional gels (NEPHGE). An acidic group of polypeptides with a molecular weight of about 40 kDa and a pI value of around 5.0 (numbered 48/59/53) were identified as antigens on Western blots probed with chronic human infection sera or vaccinated mouse sera. Polypeptides 48/49/53 from silver-stained NEPHGE gels produced antisera that were specific as demonstrated by Western blot analysis and immunoprecipitations of in vitro translation products. A cDNA clone (clone 1) from a S. mansoni adult worm pBR322 library was isolated by using cDNA probes made from size-fractionated mRNA and defined as encoding polypeptide 49 by hybridization selection of the mRNA which was in vitro translated and immunoprecipitated with specific mouse antiserum. A lambda gt 11 expression clone which contained an insert close to the full length mRNA was isolated from a S. mansoni cercariae library. The complete sequence of the mRNA was determined by sequencing the insert of this clone as well as primer extension of total RNA. The only open reading frame coding for 284 amino acids in the 1316 nucleotide sequence showed a 44.76 to 55.44% homology with the amino acid sequences of 18 different tropomyosins from various species. Computer-predicted secondary structure of schistosome tropomyosin was mainly alpha-helix which was very similar to other tropomyosins. Northern analysis showed the mRNA to be about 1.5 kb in size and detectable at much higher levels in the adult worm stage as compared to the cercariae and the egg stages. Western blot analysis likewise showed that greater amounts of tropomyosin were detected in extracts from adult worm stage as compared to extracts from cercariae and egg stages. Immunocytochemical analysis shows that tropomyosin is strongly associated with the tegument of adult worms. The restriction digestion pattern given by genomic Southern analysis suggests the existence of introns and/or multiple gene copies. Thus polypeptide 49, an immunodominant antigen, represents schistosome tropomyosin.     

Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1)

A new full-length cDNA encoding a novel protein was isolated from our human fetal brain cDNA library. The cDNA consists of 2701 bp and has a putative open reading frame encoding 131 amino acids which possesses a JAK binding site (Pro(46)-Ile-Pro(48) which is preceded by a cluster of hydrophobic residues) and is highly homologous to the leptin receptor gene-related protein (OB-RGRP). Northern blot analysis showed that this new gene is widely expressed in human tissues and radiation hybrid mapping placed the gene to human chromosome 8p21.1-8p21.2.     

Structural basis for the recognition of histone H4 by the histone-chaperone RbAp46

RbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed beta propeller structure and binds histone H4 in a groove formed between an N-terminal alpha helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.     

An immunogenic Mr 23,000 integral membrane protein of Schistosoma mansoni worms that closely resembles a human tumor-associated antigen

A Mr 23,000 Ag of the human trematode parasite, Schistosoma mansoni, has been identified by immunoscreening an adult worm cDNA library with antibody affinity purified on the Mr 23,000 to 25,000 integral membrane protein fraction of the parasite. This Ag is immunogenic in infected humans as well as in rabbits exposed to S. mansoni. The protein sequence of the Ag as deduced from cloned DNA sequences is 218 amino acids long and contains four putative transmembrane regions. Of particular significance, the Ag is strikingly similar, with respect to both amino acid sequence (36% identity) and putative domain structure to ME491, a human stage-specific melanoma-associated Ag.     

人类新基因WDR24的研究初报

WD40家族是一类结构保守、功能复杂的蛋白.目前很多研究显示该家族成员通过参与MAPK信号途径调控细胞内信号转导而影响细胞的基本生命活动.为了鉴定参与细胞生命活动的新基因,运用同源基因克隆法,通过PCR技术扩增获得一个新的人类基因WDR24, 其cDNA全长3 302 bp,2 373 bp长的开放阅读框编码由790个氨基酸残基组成的蛋白质.生物信息学分析表明,WDR24蛋白在进化上高度保守,与其他脊椎动物中的同源蛋白组成了一个功能未知的亚家族.蛋白序列分析显示其中有6个WD40重复序列和1个ERK的停泊位点D-domain.RT-PCR分析表明,该基因在所有被检测的人类胚胎组织中表达.     

The p55 subunit of Drosophila chromatin assembly factor 1 is homologous to a histone deacetylase-associated protein.

To gain a better understanding of DNA replication-coupled chromatin assembly, we have isolated the cDNA encoding the smallest (apparent molecular mass, 55 kDa; termed p55) subunit of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1), a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide comprises seven WD repeat motifs and is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. dCAF-1 was immunopurified by using affinity-purified antibodies against p55; the resulting dCAF-1 preparation possessed the four putative subunits of dCAF-1 (p180, p105, p75, and p55) and was active for DNA replication-coupled chromatin assembly. Moreover, dCAF-1 activity was specifically depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1. p55 is localized to the nucleus and is present throughout Drosophila development. Consistent with the homology between p55 and the HD1-associated RbAp48 protein, histone deacetylase activity was observed to coimmunoprecipitate specifically with p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein becomes associated with the newly assembled chromatin following DNA replication. These findings collectively suggest that p55 may function as a link between DNA replication-coupled chromatin assembly and histone modification.     

Molecular characterization and expression of a tobacco histone H1 cDNA

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steadystate mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.     

Human platelet glycoprotein IX. Characterization of cDNA and localization of the gene to chromosome 3

Overlapping cDNAs encoding human platelet glycoprotein (Gp)IX were cloned from a human erythroleukemia cell lambda gt11 library. The possibly 'full-length' cDNA of 896 base pairs (bp) includes an open reading frame (528 bp), both 5' (222 bp) and 3' (146 bp) noncoding regions, and a poly(A) tail. Translation predicts a signal peptide of 16 amino acids and a mature protein of 160 amino acids that includes a 24 amino acid leucine-rich glycoprotein (LRG) segment. Southern blot analysis suggests the presence of a single copy of the Gp IX gene, and hybridization of Gp IX cDNA to sorted human chromosomes localizes the Gp IX gene to chromosome 3.     

Fasciola hepatica: molecular cloning, nucleotide sequence, and expression of a gene encoding a polypeptide homologous to a Schistosoma mansoni fatty acid-binding protein.

Immunization of mice with an antigenic polypeptide from Fasciola hepatica adult worms and having an apparent molecular mass of 12,000 Da (Fh12) has been shown to reduce the worm burden from challenge infection with Schistosoma mansoni by more than 50%. Moreover, mice infected with S. mansoni develop antibodies to Fh12 after 5-6 weeks of infection, indicating that this Fasciola-derived antigen is a cross-reactive, cross-protective protein. A lambda gt11 F. hepatica cDNA library was constructed from poly(A)+ RNA extracted from adult worms. A cDNA encoding a cross-reactive polypeptide (Fh15) was cloned by screening the F. hepatica lambda gt11 library with a monospecific, polyclonal rabbit antiserum against pure, native Fh12. The cDNA was sequenced and the predicted amino acid sequence revealed an open reading frame encoding a 132-amino-acid protein with a predicted molecular weight of 14,700 Da. This protein has significant homology to a 14-kDa S. mansoni fatty acid-binding protein. Comparison of the protective-inducing activity of recombinant Fh15 with that of purified Fh12 against schistosomes and Fasciola is warranted.     

A conserved family of WD-40 proteins binds to the retinoblastoma protein in both plants and animals.

In mammalian cells, the retinoblastoma (RB) protein regulates G1 progression and functions through its association with various cellular proteins. Two closely related mammalian RB binding proteins, RbAp48 and RbAp46, share sequence homology with the Msi1 protein of yeast. MSI1 is a multicopy suppressor of a mutation in the IRA1 gene involved in the Ras-cAMP pathway that regulates cellular growth. Human RbAp48 is present in protein complexes involved in histone acetylation and chromatin assembly. We report the cloning of cDNAs encoding four plant RbAp48- and Msi1-like proteins: one from tomato, LeMSI1, and three from Arabidopsis. Complementation studies confirm that LeMSI1 can function as a multicopy suppressor of the yeast ira1 mutant phenotype. The LeMSI1 protein localizes to the nucleus and binds to a 65-kD protein in wild-type as well as ripening inhibitor (rin) and Neverripe (Nr) tomato fruit. LeMSI1 also binds to the human RB protein and the RB-like RRB1 protein from maize, indicating that this interaction is conserved between plants and animals.     

Complete sequence of Sipunculus nudus erythrocyte histone H2B and its gene. Identification of an N,N-dimethylproline residue at the amino-terminus

The complete amino acid sequence (122 residues) of histone H2B from erythrocytes of the marine worm Sipunculus nudus, has been established from sequence analysis of peptides generated by highly specific cleavage of the protein and from the nucleotide sequence of the encoding gene. The isolation of the H2B gene was facilitated by using a highly specific nucleotide probe, devised from amino acids 58-68 of the protein. The presence of an N,N-dimethylproline residue at the amino-terminus of the protein was established from data provided by mass spectrometry and NMR spectroscopy. This unusual post-translational modification of histone H2B generates a stable positive charge which could strongly interact with the linker DNA.     

Cloning, characterization and DNA immunization of an Onchocerca volvulus glyceraldehyde-3-phosphate dehydrogenase (Ov-GAPDH)

In the search for Onchocerca volvulus antigens possibly involved in protection against human onchocerciasis, partial amino acid sequence analysis of one of the O. volvulus antigens of the serologically identified proteins showed a close relationship to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein family. Subsequent adult worm cDNA library screening and cloning produced a clone of 1650 bp. An open reading frame spans over 1020 bp encoding for a protein of 340 amino acids with an apparent molecular weight of 38000. Comparison of the complete amino acid sequence identified this protein as a member of the GAPDH protein family. The recombinantly expressed protein shows GAPDH enzymatic activity as well as plasminogen-binding capacity. DNA sequence analysis of the corresponding gene revealed the presence of two introns. Using immunohistology Ov-GAPDH was observed in microfilariae, infective larvae, and adult male and female worms. Most striking was the labelling of the musculature of the body wall. Labelling was also observed in the pseudocoeloma cavity and in a subset of cell nuclei, suggesting additional, non-glycolytic functions of the Ov-GAPDH. Gene gun immunization with the DNA-construct in cattle led to specific humoral immune responses. Thus, the protective potential of the DNA-construct of Ov-GAPDH can be evaluated in vaccination trials using animal models such as the cattle/Onchocerca ochengi model.