人类1号染色体可变剪接与普通剪接基因同义密码子的使用分析I.同义密码子偏爱使用分析

人类1号染色体可变剪接(选择性剪接)基因344非冗余蛋白质编码序列(188183密码子)和普通剪接(非可变剪接)基因的386蛋白质编码序列(223116密码子)被用于研究人类密码子使用偏爱模式.全部密码子使用数据分析表明,人类可变剪接基因密码子的偏爱水平显著高于普通剪接基因.在人类1号染色体基因中,密码子第三位置的G C含量有很大的异质性(0.24～0.95),并且可变剪接基因密码子第三位置平均G C含量(64.66%)大于普通剪接基因(59.97%).Nc值对GC3s图显示密码子偏爱使用除了受核苷酸组成制约外,其它的因子可能也影响密码子的使用变化.此外,可变剪接基因中以G 或C结尾的密码子比普通剪接基因出现的频率高.密码子使用的差异可能是由可变剪接基因pre-mRNA特有的结构特征和多种剪接模式决定的.     

Molecular Genetic Characterization of Thyroid Dyshormonogenesis in a French Bulldog

Background

A case of congenital hypothyroidism with goiter (CHG) in a juvenile French bulldog was identified and hypothesized to be caused by dyshormonogenesis of genetic etiology.

Objectives

To describe case management, unusual phenotypic aspects, and a CHG‐causing mutation in a French bulldog.

Animals

Thyroid tissue and blood from a CHG‐affected French bulldog and 4 normal control dogs and buccal brush samples of 125 French bulldogs were studied.

Methods

Standard clinical assessment and laboratory tests were applied. Thyroid peroxidase (TPO) iodide oxidation activity was measured in vitro, and TPO protein was assessed on Western blots. Thyroid peroxidase exons and flanking splice sites were amplified from genomic DNA and sequenced. Thyroid peroxidase cDNA was amplified from thyroid RNA and sequenced.

Results

At 9 months of age, the affected dog had signs of cretinism, but near‐normal skeletal maturation. The enlarged thyroid glands exhibited noninflammatory fibrosis and aberrant follicular organization. Thyroid peroxidase activity and immunocrossreactive protein were undetectable. There was a T>C mutation of the intron 12 splice donor consensus that caused abnormally spliced mRNA, consistent with absent TPO function. The mutant allele was not observed in 125 clinically normal French bulldogs.

Conclusions

Presumptive CHG in a French bulldog with unusual clinical presentation is described. Genetic etiology was confirmed by identifying the underlying TPO mutation.     

Resistance to benzimidazole can be caused by changes in β-tubulin isoforms

The summary reports work that indicates that resistance in Rhynchosporium secalis to benzimidazole fungicides could result from substitution of the normal wild-type benA β-tubulin gene by other β-tubulin isoforms. © 1999 Society of Chemical Industry     

Purification and characterization of phospholipase A2 isoforms from the hepatopancreas of red sea bream, Pagrus major1

Two phospholipase A₂ (PLA₂) isoforms, tentatively denoted as DE-1 and DE-2 PLA₂s, were purified from the hepatopancreas of red sea bream (Pagrus major) to near homogeneity by sequential column chromatography on S-Sepharose fast flow, DEAE-Sepharose fast flow and butyl-Cellulofine, and by ion-exchange, gel-filtration and reversed-phase HPLC. The purified DE-1 and DE-2 PLA₂s both showed a single band with the apparent molecular mass of approx. 13.5 kDa by SDS-polyacrylamide gel electrophoresis, and were found to be both related to group I PLA₂ based on the N-terminal amino acid sequences. DE-1 PLA₂ had a pH optimum in the alkaline region at around pH 10 and required approximately 10 mM of Ca²⁺ and 4-10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine and phosphatidylethanolamine as substrates. DE-2 PLA₂ also had a pH optimum in the alkaline region at around pH 8-9 and required >10 mM of Ca²⁺ and approximately 6 mM of sodium deoxycholate for its maximum activity with 2 mM of phosphatidylcholine as a substrate; its enzymatic activity towards phosphatidylethanolamine was greatly inhibited by the addition of sodium deoxycholate. The results demonstrate that red sea bream hepatopancreas contains two enzymatically distinct group I PLA₂ isoforms.     

草菇TPI基因的克隆、结构及其在同核、异核菌株中的表达量

分别从不能出菇的草菇单孢菌株PYd21(基因组测序菌株)和PYd15(重测序菌株)及由PYd21、PYd15配对杂交获得可正常出菇的异核菌株H15-21中克隆测序了磷酸丙糖异构酶(TPI)基因,并对TPI基因的结构及其在3个菌株中的表达量进行了分析.结果表明:PYd21、PYd15中TPI基因的序列完全相同;转录组读段定位、双末端分析结果显示,TPI基因全长1015 bp,开放阅读框序列全长756 bp,编码251个氨基酸,有6个外显子、5个内含子;RNA加工过程中存在两种可变剪切类型和两个可变剪切位点;数字基因表达谱测序的结果表明,PYd21、PYd15和H15-21中TPI基因的表达量(TPM)分别为30.25、36.49、77.17,同时通过实时荧光定量PCR分析3个菌株中TPI基因的表达情况证实了该结果,表明草菇中TPI基因的表达表现出异核体表达的协同增效作用,预示着异核菌株中两种不同细胞核存在相互作用.     

可变剪接的表观遗传学调控机制及其在脂肪代谢中的作用研究进展

可变剪接是指从1个mRNA前体中通过不同的剪接方式产生不同的mRNA剪接异构体,并使得最终的蛋白产物表现出不同或者相互拮抗的功能和结构特性的过程.基因通过可变剪接在组织发育和疾病中起着至关重要的作用,是高等真核生物蛋白质多样性的主要来源之一.剪接过程受多种因素调控,其中表观遗传学现象是可变剪接过程中重要的影响因素,多项...     

中华蜜蜂泛素基因及其全长转录本的发掘及分析

旨在利用三代Nanopore测序技术发掘中华蜜蜂（Apis cerana cerana）泛素（ubiquitin）基因及其全长转录本。基于前期获得的中华蜜蜂工蜂4～6日龄幼虫肠道的全长转录组数据,使用BLAST工具将全长转录本的序列比对到KEGG和Nr数据库以鉴定泛素基因及其全长转录本。采用Astalavista软件分析泛素基因的可变剪接（alternative splicing,AS）事件,并通过RT-PCR加以验证。通过TAPIS pipeline软件分析泛素基因的可变多聚腺苷酸化（alternative polyadenylation,APA）位点。共鉴定到48个中华蜜蜂泛素基因和435条泛素基因相关全长转录本。共发掘到48个泛素基因的74次AS事件,包括31次内含子保留事件,19次可变3′端剪接事件,16次可变5′端剪接事件及8次外显子互斥事件。RT-PCR结果证实了3种AS事件类型的真实性。共预测到38个泛素基因含有1个及以上的APA位点,并且在APA位点的上游鉴定到多个motif,一致性序列为：KCWYTDYTMWSYGMW SCARAWCCAGAATGAYCCWYWGGHWVMWGWDRTRGC。研究结果丰富了中华蜜蜂泛素基因及其全长转录本信息,为持续深入开展相关功能研究提供参考和依据。     

花生组培苗高效嫁接技术

为解决花生野生种及转基因组培苗移栽成活率低、田间长势弱等难题，本研究对花生的嫁接技术进行了优化。以黑籽花生品种豫花0215分别在黑暗和光照条件下培养7d的实生苗为砧木，以栽培种、野生种及双二倍体的无菌组培苗为接穗，采用劈接法，以花生的下胚轴为嫁接部位进行嫁接。结果表明，花生砧木的培养方式不同，嫁接成活率差异显著，在黑暗条件下培养的砧木下胚轴达到6cm以上，平均嫁接成活率较高，达到96%以上。不同类型接穗嫁接成活率差异很大，栽培种的嫁接成活率较高，嫁接到黑暗培养的砧木上成活率达到100%。采用黑暗培养的豫花0215作砧木，大大提高了嫁接成活率和嫁接效率，该方法在花生野生种、双二倍体及转基因植株等组培苗的嫁接上获得成功，克服克服了野生种及后代材料生根困难及移栽成活率低，及组培苗移栽后长势弱等问题，值得推广。     

山羊INSR基因可变剪接体在背最长肌中的表达模式

旨在对山羊胰岛素受体（insulin receptor，INSR）基因进行可变剪接分析，研究其在背最长肌中的表达模式。本试验以简州大耳羊妊娠期胎儿（妊娠期第45、60和105天）及出生后第3天羔羊背最长肌为试验材料，基于转录组测序数据（accession number：SRR3567144）并利用Sanger测序验证，分析INSR基因的可变剪接体类型及其在背最长肌中的表达差异，同时利用在线软件对不同可变剪接体进行生物信息学分析。结果显示，在山羊背最长肌中INSR基因CDS区存在4种不同的可变剪接形式（INSR1、INSR2、INSR3和INSR4），其CDS长分别为4 110、4 146、4 113和4 149 bp，对应的氨基酸残基数分别为1 369、1 381、1 370和1 382个。山羊INSR 4个可变剪接体之间的主要区别在于Exon 11（36 bp）跳跃和Exon 15部分（3 bp）缺失，这两种可变剪接模式在牛、猪、山羊、人、绵羊和小鼠间完全一致。这些可变剪接改变了山羊INSR的Ser磷酸化位点和第二个FN3功能域，使所编码蛋白3D结构在两个区域发生明显改变。同时，各可变剪接体表达量在背最长肌发育的4个时期均无显著性差异（P>0.05），同一时期各可变剪接体表达量之间也无显著性差异（P>0.05）。INSR基因序列和剪接模式在哺乳动物中高度保守，该研究结果将为深入揭示INSR基因对动物肌肉发育的调控机制提供理论参考。