The Influence of Aerial Exposure on Sea Anemones Aulactinia veratra Mucin Genes Expression Using the RNA Sequencing

Mucin genes are the main component of mucus. The sea anemone species, Aulactinia veratra (Phylum Cnidaria) contains different types of mucin genes. In the intertidal zone, A. veratra is found to be exposed to air during the low tide and produces large quantities of mucus as an external covering. The relation between low tide and mucus secretion is still unclear, and what is the role of mucin during arial exposure is not yet investigated. This study hypothesised that the mucin genes in A. veratra would have significantly high expression in response to aerial exposure. Therefore, the aim of current study was to examine and analyses the response of A. veratra mucins in response to an experiment involving three hours of aerial exposure. To achieve this, aim the RNA-sequencing and bioinformatics analyses were used to examine the expression profile of A. veratra mucin genes in response to aerial exposure. The generated results have shown that, Mucin4-like and mucin5B-like were up-regulated in response to the three hours of aerial exposure in A. veratra. This finding shows a significant role of mucin5B-like and mucin4-like genes in response to air stress at low tide. The data generated from this study could be used in conjunction with future mucin gene studies of sea anemones and other cnidarians to compare A. veratra mucin gene expression results across time, and to extend our understanding of mucin stress response in this phylum.

Identification and characterization of adult alpha-and beta-globin genes and their genomic arrangement in Pseudosciaena crocea

The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3 ＇-end （ 3 ＇-RACE）. The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA stop codon at Position 469 and the AATAAA polyadenylation signal at Position 560, which codifies 145 amino acids. The entire open reading frame of the β-globin gene is 447 bp long, which encodes 148 amino acids. Amino acid identity of the α- globin or β-globin gene compared with those reported in other fish species, ranged from 31.9% to 76.4%. When comparing with human α- and β-globins, three important alterations in the structural regions can be noted： ct39 Thr→Gln, α113 His→Tyr and β117 His→Lys. The α-globin has a unique inserted amino acid residue in the 47th position. To understand the process of globin gene duplication and identify the regulatory elements present in the intergenic and intragenic regions of globin genes, the genomic arrangement of α- and β-globin genes was investigated. The results showed that the orientation of the two genes was head-to-head relative to each other. The intergenic region between the translation initiation codons of the linked α- and β-globin genes contains classical promoter elements and the length of it is much shorter than that reported in other fish.

Trans-acting factors from the human fetal liver bindingto the human ε-globin gene silencer

The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp～-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin this silencer element was identified. Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.

Chromatin-binding in vivo of the erythroid kruppel-like factor,EKLF,in the murine globin loci

EKLF 是一 erythroid 特定，锌为在在权威的系的 erythroid 房间的基因的哺乳动物的贝它滴的激活的包含手指的抄写因素必需品。我们准备了对西方的弄污和免疫降水(IP ) 质量合适的 polyclonal 反老鼠 EKLF 抗体，并且使用了它在老鼠 erythroid 开发期间定义 EKLF 蛋白质的表示模式。我们也为 chromatin-immunoprecipitation (薄片) 使用了这抗体试金。EKLF 被发现以一种 DMSO 可诱导的方式在老鼠 erythroleukemia 房间(MEL ) 在老鼠 beta-major-globin 倡导者和 beta-LCR 的 HS2 地点在 vivo 绑。象三另外的蛋白质一样的 EKLF 的 导致DMSO 的绑定，也就是 RNA 聚合酶 II ，乙酰 ated 嘘一 H3 ，和 methylated 嘘一 H3 ，没在 CB3 被废除，但是显著地降低了，一根 导出MEL 的房间线与 p45/NF-E2 空表示，在 loci 为哺乳动物的滴的激活需要的一个 充实erythroid 的因素。有趣地，在 vivo 的 EKLF 的绑定也在地点在鼠标象 alpha 一样滴被检测，在成年高山哈在倡导者和它的远在上游的规章的元素 alpha-MRE (HS26 ) 的滴。这研究提供直接证据为在在在基因的像贝它的滴聚类的老鼠的主要规章的元素的 vivo EKLF 有约束力数据也在基因规定在哺乳动物的滴关于 EKLF 染色质相互作用的角色有有趣的含意。

PREDOMINANT EXPRESSION OF HUMAN Aγ-IN CONTRAST WITH β-GLOBIN GENE IN MEL CELLS TRANSFECTED WITH THE CONSTRUCT μLCRAγψβδ

ＩＮＴＲＯＤＵＣＴＩＯＮＴｈｅｈｕｍａｎβ－ｇｌｏｂｉｎｇｅｎｅｃｌｕｓｔｅｒｃｏｎｓｉｓｔｓｏｆｆｉｖｅｆｕｎｃｔｉｏｎａｌｇｅｎｅｓａｓｗｅｌａｓｏｎｅｐｓｅｕｄｏｇｅｎｅｉｎｔｈｅｏｒｄｅｒｏｆ５＇ε，Ｇγ，Ａγ，ψβ，δ，βａｎｄｄｉｓｐｌａ...

Application of α-globin 3′hypervariable region to gene diagnosis of adult polycystic kidney disease

Seventy-four members from 9 adult polycystic kidney disease(APKD)familieswere analysed with Southern blot and 3′hypervariable region/Pvu Ⅱ,3′HVR/Bgl ⅡRFLPs.The results showed that 3′ HVR fragments were highly polymorphic.Theheterozygote frequencies of Pvu Ⅱ,Bgl Ⅱ and PvuⅡ+Bg1Ⅱ fragments were 92%,84.7%and 98.6%,respectively.The maximum lod score for linkage between 3′HVR and APKDwas 9.71 at a recombination fraction 0.045.We successfully applied 3′HVR probe to thegene diagnosis of 17 symptomatic patients and 7 patients in the presymptomatic stage.

乌鳢globin基因家族的鉴定及在鳃和鳃上器官中的表达模式分析

为了探究珠蛋白(Globin)在具有耐低氧和空气呼吸能力的乌鳢(Channa argus)中发挥的作用,本文对乌鳢的globin基因家族进行研究。在基因组中鉴定得到18个globin基因,分属于Hb、Mb、Ngb和Cygb四个亚家族,其中Hb亚家族拷贝数最多,达14个,其余各亚家族仅有1~2个拷贝。系统进化分析表明,乌鳢globin基因与硬骨鱼类聚为一枝,在进化上较为保守。对乌鳢globin基因结构分析得到4种保守结构域和5个motif。利用转录组测序技术对仔稚鱼(受精后3~8 d)和幼鱼(4月龄)的鳃和鳃上器官中globin基因进行表达量检测。在仔稚鱼中,hbae1(包括hbae1.1、hbae1.2和hbae1.3)、hbb2(包括hbb2.1、hbb2.2和hbb2.4)和hbaa的表达量在鳃上器官发育过程中呈显著升高趋势,而hba1和hbb1的表达量则显著降低。在幼鱼中,在空气暴露胁迫后,hbb2.2和cygb的表达量在鳃和鳃上器官中均发生显著变化,hbae1.1、hbae1.2和hbba的表达量在鳃中发生显著变化,hbae1.3和hbb2.1的表达量在鳃上器官中发生显著变化。这些在鳃上器官发育过程中和空气暴露胁迫中表达量出现显著变化的globin基因可能在乌鳢空气呼吸及低氧胁迫中发挥了生物学功能。

Imprinting, methylation, and expression characterization of the maize ETHYLENE-INSENSITIVE 2-like gene

The endosperm plays essential roles in embryogenesis and seed germination and provides abundant resources for human food and industrial products. Identification of genes regulating the development of the endosperm and elucidation of their functions is of great importance for maize genetics and breeding. This study showed that the genespecific imprinted gene, ETHYLENE-INSENSITIVE 2-like(EIN2-like), is maternally expressed in both endosperm and embryo. The maternally expressed pattern was maintained throughout later seed developmental stages. Bisulfite sequencing using DNA obtained from hybrid endosperm tissues showed that the upstream regions of the alleles of EIN2-like were highly methylated at symmetrical sites(CG and CHG). A differentially methylated region in the upstream part of the maternal allele of EIN2-like was identified and found to be hypomethylated. Expression analysis showed that EIN2-like was highly expressed in the maize endosperm as well as at different stages of cell differentiation(8–12 days after pollination) in the hybrid endosperm. These results suggest that the maternally expressed gene EIN2-like may play crucial roles in the regulation of seed development.

Identification of the development stage-specific factors in mouse fetal liver binding to the human β-globin gene promoter

In order to elucidate the molecular mechanisms of globin gene expression during embryonic development, the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared. By using DNase I footprinting and gel mobility shift assays, the binding of protein factors in these extracts to the human βglobin promoter was analyzed. The differences in the binding patterns of protein factors during development were observed. An erythroid-specific and stage-specific nuclear protein in the nuclear extract from d 18 mouse fetal liver was identified, which can bind to the sequence (from -66bp to -90bp) of human β-globin promoter. We therefore speculate that the function of this cis-acting element may be similar to stage selector element (SSE) in chieken βA- promoter.

ISOBUTYRAMIDE ACTIVATES TRANSCRIPTION OF HUMAN FETAL γ-AND MURINE EMBRYONIC εy-GLOBIN GENES

Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globin gene expression and to test cell toxicity ofthe drug.Methods. MEL cells were transfected with the recombinant construct μLCRAγψβδβand the stable transformants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rabbit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection assay. The hematological toxicity and electrolyte toxicity ofthe drug were tested.Results. An inducible and dose dependent expression of the human γ , β and mouse α globin gene was observed in the transfected MEL cells. The induction of the human γ globin gene is significant stronger than that of the β globin gene. With 2.5～5 mmol/L isobutyramide, the induction of the human γ globin gene is even more effective than that of mouse α globin gene. After a 15 day injection under the doses of 500～900mg·kg-1·d-1, the level of the mouse embryonic εy globin mRNA could be significantly induced up to 3～4 fold of that of uninjected controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected with different doses of isobutyramide at the interval of 24 hours for 2～4 weeks, were generally within the normal range. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+, Na+, Cl-and CO2 were all within normal range and serum ionic osmotic pressure remained stable as well. Conclusion. Our results suggested that isobutyramide is a weak inducer ofcell differentiation, but it can selectively activate transcription of human γ globin gene at a certain degree, and it can act on early stages of erythroid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have no hematological toxicity. Our results have further proved the potential value of isobutyramide in treatment of β-thalassemia and sickle cell disease.

Identification of a NF-кB site in the negative regulatory element (ε-NRAII) of human ε-globin gene and its binding protein NF-кB p50 in the nuclei of K562 cells

The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5’ flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp～ -2902bp, termed ε-NRAII) 5’ to the cap site of this gene. NRF DNA fragment (-3010bp～ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.

Proteins binding to the 5'-flanking regulatory elements of the human β-globin gene^1

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene,two negative control regions(NCR1,-610to-490 bp;NCR2,-338,to-233bp),was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in art for the silence of β-glolbin gene in the embryonic and fetal stages.

Co-Inheritance of Beta &Delta-Globin Gene (HbYialousa) Mutations in an Iranian <i>β</i>-Thalassemia Carrier

Introduction: Beta-thalassemia is characterized by absence or reduced synthesis of the β-globin. Carriers of β-thalas- semia, typically have microcytic hypochromic anemia and elevated hemoglobin HbA2 and normal HbF level. On the other hand carriers of severe alpha-thalassemia also have similar CBC parameters to that of β-thalassemia with normal HbA2 level. Co-presence of mutations in the β-globin and delta-globin genes (point mutations or deletions) usually give normal HbA2 and elevated HbF level. We report a β-thal carrier with normal level of HbA2 and increased level of HbF who had a point mutation in CD39 on the beta-globin gene and a point mutation in CD27 on the δ-globin gene named Hb-Yialousa. Materials & Methods: An individual with low hematological indices, normal HbA2 and elevated HbF was referred to our center as routine premarital screening program. Mutations in the β-globin and δ-globin genes were screened using ARMS and sequencing methods. Results: The mutation in β- and δ-globin genes were identified as CD39 and CD27 (HbYialousa) respectively. No point mutation or deletion in α-globin gene was identified. Discussion: We showed that normal HBA2 with elevated HbF level is due to co-inheritance of delta-globin gene mutation with mutation in the β-globin gene. When screening for β-thalassemia, one has to either rule out presence of α-globin gene mutation of mutation in the delta-globin gene.

The 5'-flanking cis-acting elements of the human ε-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.

A DNA-binding protein factor in K562 nuclear extract interacts with positive control region (PCR) in the 5'flanking sequence of human β-globin gene

It has been known that there are at least three regulatory regions (NCR1. NCR2 and PCR) in the 5'-flanking sequence (from -610 bp to +1 bp) of human β-glohin geneand that the function of PCR is unique to the human erythroleukemia (Ksfi2) ceils. Here we have detected a DNA-binding protein factor (termed NFEa) in K562 ceils. which can bind specifically to the PCR of human β-globin gene. The sequence of the binding site is 5'ACTGATG3' (between -222 bp and -216 bp). The NFEa is erythroidspecific and perhaps specific for K562 cells. It seemed that this factor differed from the erythroid-specific transcriptional factor (NFE-1) ,nsing competition assay. The presence of the NFEa further supported that the funciton of the cis-acting element PCR was specitic for K562 cells. and helps us to understand the mechauism of the regulation of the expression of lmman β-globin gene in the human K562 cells.

Studies on DNA-protein interactions in the upstream regulatory region of the human ε-globin gene promoter

The erythroid- and developmental stage-specific expression of the human ε-globin gene is controlled, in part,by the 5’-flanking DNA sequence of this gene. In the present study, we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ε-globin gene during development. Using gel mobility shift assays and DNasel footprinting assays, a nuclear protein factor (termed ε-SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified. It could specifically bind to the positive control region (between -535 and -453bp) of the human ε-globin gene. We speculated that the E-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ε-R1) in the nuclear extract from mouse fetal liver at d 18 of gestation, which could strongly bind to the silencer region (between -392 and -177bp) of this gene. Therefore, we speculated that the ε-R1 might be an erythroid- and developmental stagespecific repressor. Our data suggest that both ε-SSF1 and ε-R1 might play important roles in developmental regulation of the human ε-globin gene expression during the early embryonic life. On the other hand, we observed that the binding patterns of nuclear proteins from three cell lines (K562, HEL and Raji) to these regulatory regions were partially different. These results suggest that different trans-acting factors in K562, HEL and Raji cells might be responsible for activating or silencing the human ε-globin gene in three different cell lines.

HBA2:c.2T>C和HBA2:c.2delT两例罕见突变引起血红蛋白H病家系分析

目的:分别对HBA2:c.2T>C和HBA2:c.2delT两种罕见HBA2基因起始密码子突变复合东南亚型α-地贫的血红蛋白H病病例及其家系成员进行致病基因分析,了解HBA2:c.2T>C和HBA2:c.2delT突变与临床表型的关系。方法:采集家系成员外周血进行血细胞分析及毛细管电泳血红蛋白分析,缺口PCR(Gap-PCR)、反向点杂交法(RDB)检测ɑ-地贫基因常见类型突变,Sanger测序法对HBA1和HBA2基因序列进行分析。结果:检测出两个先证者基因型分别为--SEA/αα复合HBA2:c.2T>C和--SEA/αα复合HBA2:c.2delT,家系成员中检出HBA2:c.2T>C/WT和HBA2:c.2delT/WT,均表现为小细胞低色素性贫血。结论:HBA2:c.2T>C和HBA2:c.2delT为杂合突变时机体可出现静止型α-地贫的表型,当其复合轻型α-地贫时可使机体出现血红蛋白H病的临床表现,本研究为遗传咨询提供依据。

^(A)γ-珠蛋白基因启动子区域-114~-102缺失突变导致非缺失型遗传性持续性胎儿血红蛋白综合征的研究

目的:研究^(A)γ-珠蛋白基因启动子区域-114~-102缺失突变病例的基因突变特点、血液学表型及临床表型。方法:选取2022年3月至2023年12月在广西医科大学第一附属医院检查地中海贫血的病例4例。血红蛋白(Hb)分析仪和血细胞分析仪检测全血样本,分析Hb和血常规。Sanger测序法与荧光PCR熔解曲线法检测γ-珠蛋白基因启动子区突变和β-珠蛋白基因突变。结果:共检出^(A)γ-114~-102缺失杂合子4例,Hb分析显示血红蛋白F(Hb F)水平为11.2%~37.8%,血红蛋白A2(Hb A2)水平为2.0%~3.3%,血常规分析显示Hb水平为67~114 g/L,红细胞计数(RBC)为(2.33~4.1)×10^(12)/L,平均红细胞体积(MCV)为84.02~91.9 fL,平均红细胞血红蛋白(MCH)为27.8~29.7 pg。γ-珠蛋白基因及β-珠蛋白基因突变检测结果表明:4例病例含有^(A)γ-114~-102缺失杂合子,其中2例合并有^(G)γ-158C>T突变杂合子;4例病例均合并有β-珠蛋白基因突变,基因型分别为Codon 41-42(-TTCT)杂合子突变、Codon 41-42(-TTCT)纯合子突变、IVS-Ⅱ-654(C>T)复合Codon 41-42(-TTCT)突变和Codon17(A>T)复合Codon 71-72(+A)突变。结论:首次在中国人群中发现^(A)γ-114~-102缺失杂合子,且复合β-地中海贫血的病例,临床表现为轻度或中度贫血。^(A)γ-114~-102缺失突变导致非缺失型遗传性持续性胎儿血红蛋白综合征(nd-HPFH),能够减轻β-地中海贫血患者的贫血程度。

Diversity of the T4-like Myoviruses Community in Response to Salinity in Saline Lakes of the Qinghai-Tibetan Plateau

1 Introduction Viruses are the most abundant biological entities on Earth.They can influence the succession of individual microbial populations,biogeochemical cycles of C/N and,ultimately,microbial community structure through killing