Evolutionary Origin and Molecular Biology of the Melanoma-Inducing Oncogene of Xiphophorus

Melanoma formation in platyfish/swordtail hybrids of genus Xiphophorus is due to overexpression of the receptor tyrosine kinase oncogene Xmrk. This gene is the molecular equivalent to the Tu-locus of platyfish, formerly identified by Mendelian genetics. The supposed evolutionary origin of the Xmrk oncogene is a nonhomologous recombination event in the 5’region of the corresponding Xmrk protooncogene with an anonymous sequence, D. This event led to a gene duplication of Xmrk, whereby the new copy obtained a novel promoter derived from D. Inactivity of this promoter in parental fish warrants lack of tumorigenicity of the Xmrk oncogene in wild playfish. In hybrids, however, the promoter is active. This leads to the pigment cell transforming overexpression of Xmrk.     

剑尾鱼杂交种黑色素瘤的形成和病理观察

目的通过剑尾鱼属内鱼类的杂交,观察黑色素瘤在其杂交后代的形成情况。方法白体剑尾鱼(♀)与黑体剑尾鱼(♂)杂交,子一代(F1)和子二代(F2)分别自交,观察其后代外部特征变化和黑色素瘤形成的情况;对黑色素瘤进行组织病理观察。结果剑尾鱼杂交种的F2代开始出现性状分离,F3中有较高的黑色素瘤发生率,为20.5%。HE染色和Lillie黑色素染色证实增生组织为黑色素瘤。结论通过不同剑尾鱼的杂交和自交,后代有黑色素瘤形成,可望进行黑色素瘤模型的构建。     

Human Phenol SulfotransferaseSTP2Gene: Molecular Cloning, Structural Characterization, and Chromosomal Localization

Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of “simple” planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs,STP2,as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans.STP2spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of mostSTP2exon–intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans,STM;a rat PST gene; a human estrogen ST (EST) gene,STE;and a guinea pig EST gene. The two initialSTP2exons, IA and IB, were identified by performing 5′-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5′-untranslated region sequences. The two apparent 5′-flanking regions of theSTP2gene, regions flanking exons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements.STP2was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization ofSTP2will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissue.     

癌基因iASPP的克隆、表达与纯化

iASPP是新近发现的高度保守的p53相关基因,其蛋白产物定位于细胞核内,具有结合NFκBp65亚基和p53功能,进而抑制NFκB的转录调节和p53对凋亡的调节功能。利用RTPCR方法从人白血病细胞系U937中克隆出癌基因iASPP,将其克隆至原核表达载体pET28a(+)中,成功构建iASPP表达载体PIAF,重组质粒读码框和序列与预期一致。在IPTG诱导下,重组载体大肠杆菌Rosetta(DE3)菌株,表达产物经SDSPAGE和Westernblot方法分析证实系iASPP融合蛋白。利用Ni离子鳌合层析的方法纯化iASPP融合蛋白,经SDSPAGE鉴定其纯度超过80%。     

Molecular Cloning, Expression, and In Silico Structural Analysis of Guinea Pig IL-17

Interleukin-17A (IL-17A) is a potent proinflammatory cytokine and the signature cytokine of Th17 cells, a subset which is involved in cytokine and chemokine production, neutrophil recruitment, promotion of T cell priming, and antibody production. IL-17 may play an important role in tuberculosis and other infectious diseases. In preparation for investigating its role in the highly relevant guinea pig model of pulmonary tuberculosis, we cloned guinea pig IL-17A for the first time. The complete coding sequence of the guinea pig IL-17A gene (477 nucleotides; 159 amino acids) was subcloned into a prokaryotic expression vector (pET-30a) resulting in the expression of a 17 kDa recombinant guinea pig IL-17A protein which was confirmed by mass spectrometry analysis. Homology modeling of guinea pig IL-17A revealed that the three-dimensional structure resembles that of human IL-17A. The secondary structure predicted for this protein showed the presence of one extra helix in the N-terminal region. The expression profile of IL-17A was analyzed quantitatively in spleen, lymph node, and lung cells from BCG-vaccinated guinea pigs by real-time PCR. The guinea pig IL-17A cDNA and its recombinant protein will serve as valuable tools for molecular and immunological studies in the guinea pig model of pulmonary TB and other human diseases.     

产黄青霉谷胱甘肽S-转移酶基因PcgstA的克隆与鉴定

从青霉素工业生产菌产黄青霉(Penicilliumchrysogenum)中首次克隆了一个谷胱甘肽S-转移酶(GST)基因,定名为PcgstA.该基因的开放阅读框长840bp,含有两个内含子,编码一个238氨基酸残基的蛋白质.其推断的氨基酸序列与一些已经鉴定的丝状真菌GST具有50%左右的序列一致性.PcgstA的完整编码区经RT-PCR扩增、验证,插入原核表达载体pET11a,转化大肠杆菌BL21(DE3)-RP菌株,表达得到重组PcGSTA蛋白.酶活测定证实,重组PcGSTA具有GST活性,其对底物CDNB(1-chloro-2,4-dinitrobenzene)的比活为(0.159±0.031)μmol/(min·mg).利用TaqMan探针法,对PcgstA的表达情况进行了比较.结果表明,在添加了侧链前体苯乙酸的青霉素生产培养基中,PcgstA的表达水平和在不含苯乙酸培养基中的表达相比明显下调,显示了该基因与苯乙酸代谢的关系.     

Characterization, Molecular Cloning, and Differential Expression Analysis of Laccase Genes from the Edible Mushroom Lentinula edodes

The effect of different substrates and various developmental stages (mycelium growth, primordium appearance, and fruiting-body formation) on laccase production in the edible mushroom Lentinula edodes was studied. The cap of the mature mushroom showed the highest laccase activity, and laccase activity was not stimulated by some well-known laccase inducers or sawdust. For our molecular studies, two genomic DNA sequences, representing allelic variants of the L. edodes lac1 gene, were isolated, and DNA sequence analysis demonstrated that lac1 encodes a putative polypeptide of 526 amino acids which is interrupted by 13 introns. The two allelic genes differ at 95 nucleotides, which results in seven amino acid differences in the encoded protein. The copper-binding domains found in other laccase enzymes are conserved in the L. edodes Lac1 proteins. A fragment of a second laccase gene (lac2) was also isolated, and competitive PCR showed that expression of lac1 and lac2 genes was different under various conditions. Our results suggest that laccases may play a role in the morphogenesis of the mushroom. To our knowledge, this is the first report on the cloning of genes involved in lignocellulose degradation in this economically important edible fungus.     

Purification,Characterization, and Molecular Cloning of Tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.     

小麦3个NAC转录因子基因克隆与功能分析

NAC类转录因子参与植物基因在不同条件、不同发育期的表达调控,在植物的发育、生长及对外界的各种生物和非生物因子的胁迫应答中起关键的调控作用。本研究对非亲和条锈菌小种CYR32侵染诱导的抗条锈病基因Yr5近等基因系Taichung29*6/Yr5的cDNA文库进行筛选及同源克隆,获得了小麦3个NAC类转录因子的cDNA序列。序列分析结果表明,这3个转录因子都具有DNA结合结构域,即NAC结构域,且氨基酸序列在该结构域的A、B、C、D和E5个亚区高度保守;同时发现这3个NAC类转录因子都有核定位信号及相关的转录调控功能区域。通过系统进化树分析,发现其中之一的TaNAC1属于NAC转录因子家族第Ⅰ组的NAP亚组,TaNAC3属于ATAF1亚组,TaNAC5属于NAM亚组;根据系统进化树和相关基因的功能分析,我们推测小麦转录因子TaNAC1、TaNAC3和TaNAC5可能参与植物生长发育调控或对生物、非生物胁迫作出的应答反应。     

Molecular Cloning,Characterization and Sequence Analysis of KCNQ4 in Large Odorous Frog,Odorrana graminea

Acoustic communication is essential for anuran survival and reproduction, and masking background noise can affect the effective acoustic communication. The larger odorous frog(Odorrana graminea) inhabits noise montane streams, and it has shown an ultrasound communication adaptation. However, the molecular mechanism underlying their ultrasonic hearing adaptation remains unknown. To characterize and investigate the molecular characteristics and evolution of the high-frequency hearing-sensitive gene(KCNQ4) in O. graminea, termed as OgKCNQ4, the rapid amplification of cDNA ends(RACE) was performed to amplify the cDNA of OgKCNQ4. Different bioinformatics analyses were used to investigate the molecular characteristics. Multiple nucleotide and amino acid sequence alignment were conducted, and phylogenies were reconstructed under the maximum likelihood and Bayesian approaches. The full-length cDNA of OgKCNQ4 was 2065 bp, and the open reading frame(ORF) was 2046 bp encoding for a putative protein with 681 amino acids. The relative molecular weight of OgKCNQ4 was 76.453 kD and the putative PI was 9.69. Secondary structure prediction analyses suggested 42.29% alpha helixes and 43.76% random coils in OgKCNQ4. Gene homology and Phylogenetic analyses revealed the closest relationship between OgKCNQ4 and KCNQ4 of Nanorana parkeri with 96.9% similarity and 95.0% identity. We first determined the full-length cDNA of OgKCNQ4 and the results here could provide foundations for further study on the evolution of KCNQ4 and its relationship to ultrasonic communication in amphibians.     

Molecular Cloning,Functional Characterization,and Evolutionary Analysis of Vitamin D Receptors Isolated from Basal Vertebrates

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC₅₀ values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D₃ acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D₃ throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.     

核桃JrCBF基因的克隆与表达和单核苷酸多态性分析

根据CBF基因氨基酸保守序列设计简并引物,运用cDNA末端快速扩增(RACE)技术克隆核桃JrCBF基因cDNA全长序列。用实时荧光定量PCR分析JrCBF基因在低温胁迫下的表达模式,并分析JrCBF基因的单核苷酸多态性。结果获得长度为879 bp的CBF基因cDNA全长序列,编码214个氨基酸,命名为JrCBF;低温能诱导JrCBF基因的表达,4℃处理2 h后表达量开始增加,8 h后达到最大值;自然越冬条件下,JrCBF基因在花芽中表达量呈现先上升后下降的趋势,在寒冬时期(1月份)表达量最高;单核苷酸多态性分析JrCBF基因序列中有28个SNPs位点和7个Indels标记,存在2个突变热点区;单倍型分析显示15份材料可分为9个单倍型,单倍型多样性为0.9238。本研究为通过基因工程手段培育抗寒核桃品种和分子标记辅助育种提供了帮助。     

可溶性组织因子突变体的基因构建、表达和性质

血浆中组织因子 (TF)的过量表达与许多病理过程密切相关。TF抑制物有可能防治这些疾病。设计了两种可溶性组织因子 (sTF)的突变体 (MCsTF和MFsTF) ,突变了协同催化的功能域 ,保留与因子VII/VIIa结合的功能域 ,使突变体竞争性抑制野生型TF的功能。用PCR的方法 ,对可溶性组织因子cDNA基因进行了点突变 ,并实现了在大肠杆菌中高效表达。rsTF、rMCsTF和rMFsTF的促凝活性研究表明 ,rMFsTF的激活X因子活性和促凝血作用相当于rsTF的 10 % ,而rMCsTF几乎完全失去了激活X因子活性和促凝血作用。rMCsTF和rMFsTF与VII/VIIa因子形成复合物对激活X因子的催化特异常数 (kcat/Km)分别是FVII/VIIa·rsTF的 2 .0 %和 3.7% ,也说明突变体与VII/VIIa形成的复合物对激活X因子催化活性显著降低。rMCsTF和rMFsTF对rsTF活性抑制动力学研究及体外活性研究表明 ,两种突变体均有抑制rsTF活性和抑制兔脑粉的促凝活性作用 ,抑制作用呈量效关系