Allelic Polymorphism,Gene Duplication and Balancing Selection of MHC Class ⅡB Genes in the Omei Treefrog(Rhacophorus omeimontis)

The worldwide declines in amphibian populations have largely been caused by infectious fungi and bacteria. Given that vertebrate immunity against these extracellular pathogens is primarily functioned by the major histocompatibility complex(MHC) class Ⅱ molecules, the characterization and the evolution of amphibian MHC class Ⅱ genes have attracted increasing attention. The polymorphism of MHC class Ⅱ genes was found to be correlated with susceptibility to fungal pathogens in many amphibian species, suggesting the importance of studies on MHC class Ⅱ genes for amphibians. However, such studies on MHC class Ⅱ gene evolution have rarely been conducted on amphibians in China. In this study, we chose Omei treefrog(Rhacophorus omeimontis), which lived moist environments easy for breeding bacteria, to study the polymorphism of its MHC class Ⅱ genes and the underlying evolutionary mechanisms. We amplified the entire MHC class ⅡB exon 2 sequence in the R. omeimontis using newly designed primers. We detected 102 putative alleles in 146 individuals. The number of alleles per individual ranged from one to seven, indicating that there are at least four loci containing MHC class ⅡB genes in R. omeimontis. The allelic polymorphism estimated from the 102 alleles in R. omeimontis was not high compared to that estimated in other anuran species. No significant gene recombination was detected in the 102 MHC class ⅡB exon 2 sequences. In contrast, both gene duplication and balancing selection greatly contributed to the variability in MHC class ⅡB exon 2 sequences of R. omeimontis. This study lays the groundwork for the future researches to comprehensively analyze the evolution of amphibian MHC genes and to assess the role of MHC gene polymorphisms in resistance against extracellular pathogens for amphibians in China.     

SLA的分型与检测

传统的强调抗原多态性的SLA分型方法主要是血清学、细胞学和生物化学的方法,随着分子生物学技术发展,各具特点的SLA分型技术不断涌现,如PCR-RFLP法、PCR-SSCP法、微卫星（MS）法、DNA序列的测定等,本文基于强调SLA的抗原多态性的分型和强调抗原保守性的功能学上的新分型技术（如SLA超型和超基序）进行了详细探讨,比较了各类方法的优缺点,指明了未来SLA分型的发展趋势。此外本文还指出了现行参考教材血清学SLA单倍型的编写错误以及重点强调了SLAⅡ-DQB基因的准确分型技术必须借鉴于克隆测序。Abstract：Traditionally the cluster of swine leukocyte antigen(SLA) was typed by serological, cytological and biochemical methods. Many special molecular typing methods have been developed with the progress of molecular biological technology, such as PCR-RFLP, PCR-SSCP , MS and DNA sequencing. Here we discussed the advantages and disadvantages of each method based on the polymorphic and conservative (from the functional aspect, such as supertype and supermotif) characteristics of SLA, and illustrated the development of typing for SLA in the future. In addition, we pointed out the editorial mistakes about the serological haplotye of SLA in reference book and emphasized that the accurate polymorphism of SLA-DQB gene must be based on the cloning sequencing.     

Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation

To lay background for studying rejection mechanisms in xenotransplantation and developing the strate-gies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced. The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors. All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their ac-cession numbers AY102467- AY102472 from GenBank. This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A^*0201) are better than those between pigs and mice (H-2D^b/H-2K^b). The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules. The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted. A functional test indicated that the human T cells specific for the prokaryotically expressed SLA Plprotein could respond quite well in vitro to the class I-positive swine chon-drocytes and PBMCs in presence of human APCs. This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens.     

MHC Class I Exon 4 in the Multiocellated Racerunners （Eremias multiocellata）： Polymorphism,Duplication and Selection

The major histocompatibility complex （MHC） is a dynamic genetic region with an essential role in the adaptive immunity of jawed vertebrates. The MHC polymorphism is affected by many processes such as birth-and- death evolution, gene conversion, and concerted evolution. Studies investigating the evolution of MHC class I genes have been biased toward a few particular taxa and model species. However, the investigation of this region in nonavian reptiles is still in its infancy. We present the first characterization of MHC class I genes in a species from the family Lacertidae. We assessed genetic diversity and a role of selection in shaping the diversity of MHC class I exon 4 among 37 individuals of Eremias multiocellata from a population in Lanzhou, China. We generated 67 distinct DNA sequences using cloning and sequencing methods, and identified 36 putative functional variants as well as two putative pseudogene-variants. We found the number of variants within an individual varying between two and seven, indicating that there are at least four MHC class I loci in this species. Gene duplication plays a role in increasing copy numbers of MHC genes and allelic diversity in this species. The class I exon 4 sequences are characteristic of low nucleotide diversity. No signal of recombination is detected, but purifying selection is detected in β2-microglobulin interaction sites and some other silent sites outside of the function-constraint regions. Certain identical alleles are shared by Eremias multiocellata and E. przewalskii and E. brenchleyi, suggesting trans-species polymorphism. The data are compatible with a birth-and-death model of evolution.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

中国广东汉族人群核苷酸修复基因hMTH1 遗传多态性研究

为研究中国南方汉族人群核苷酸修复基因hMTH1遗传多态性,应用聚合酶链反应-单链构象多态性技术检测172名健康人外周血白细胞hMTH1基因启动子及全部5个外显子多态性,并进行DNA测序。结果发现hMTH1基因启动子及外显子1序列保守,未见突变;外显子2第73位碱基存在T→C杂合型突变,基因型TT和TC频率分别为93.02%、6.98%,等位基因T和C频率分别为96.51%、3.49％;外显子3第45位遗传密码存在T→C杂合型突变,基因型TT和TC频率分别为95.35%、4.65%,等位基因T和C频率分别为97.67%、2.33%,该多态性为首次发现;外显子4第83位遗传密码存在G→A杂合型突变,基因型GG和GA频率分别为89.53%、10.47%,等位基因G和A频率分别为94.77%、5.23％;外显子5第119位氨基酸遗传密码存在C→T杂合型突变,基因型CC和CT频率分别为95.93%、4.07%,等位基因C和T频率分别为97.97%、2.03％。Abstract: In order to study the genetic polymorphisms of nucleotide repair gene hMTH1 in southern Chinese Han population, the polymorphisms of the gene’s promoter and its five exons among peripheral blood lymphocytes of 172 Chinese Han people were analyzed with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. The sequences of the promoter and exon 1 of hMTH1 gene were conserved. A T to C polymorphism was detected at the 73th base in exon2. The genotype frequencies of TT and TC were 93.02% and 6.98%, respectively. The allelic frequencies of T and C were 96.51% and 3.49％, respectively. A T to C polymorphism was detected at codon 45 in exon3, which was first reported. The genotype frequencies of TT and TC were 95.35% and 4.65%, respectively. The allelic frequencies of T and C were 97.67% and 2.33%, respectively. A G to A polymorphism was detected at codon 83 in exon4. The genotype frequencies of GG and GA were 89.53% and 10.47%, respectively. The allelic frequencies of G and A were 94.77% and 5.23％, respectively. A C to T polymorphism was detected at codon 119 in exon5. The genotype frequencies of CC and CT were 95.93% and 4.07%, respectively. The allelic frequencies of C and T were 97.97% and 2.03％, respectively.     

Expression of two soybean resistance gene candidates shows divergence of paralogous single-copy genes

The cloning of several plant genes directly involved in triggering a disease resistance response has shown that numerous resistance genes in the nucleotide binding site (NBS)/leucine-rich repeat (LRR) class have similar conserved amino acid sequences. In this study, we used a short soybean DNA sequence, previously cloned based on its conserved NBS, as a probe to identify full-length resistance gene candidates. Two homologous, but genetically independent genes were identified. One gene maps to the soybean molecular linkage group (MLG) F and a second is coded on MLG E. The first gene contains a 3,279 nucleotide open reading frame (ORF) sequence and possesses all the functional motifs characteristic of previously cloned NBS/LRR resistance genes. The N-terminal sequence of the deduced gene product is highly characteristic of other resistance genes in the subgroup of NBS/LRR genes which show homology to the Toll/Interleukin-1 receptor genes. The C-terminal region is somewhat more divergent as seen in other cloned disease resistance genes. This region of the F-linked gene contains an LRR region that is characterized by two alternatively spliced products which produce gene products with either a four-repeat or a ten-repeat LRR. The second cloned gene that maps to soybean MLG E contains 1,565 nucleotides of ORF in the N-terminal domain. Despite strong homology, however, the 3′ region of this gene contains several in-frame stop codons and apparent frame shifts compared to the F-linked gene, suggesting that its functionality as a disease resistance gene is questionable. These two disease resistance gene candidates are shown to be closely related to one another and to the members of the NBS/LRR class of disease resistance genes. Received: 29 November 1999 / Accepted: 22 December 1999     

多浪羊MHC-DRB3基因座的PCR-RFLP多态性分析

主要组织相容性复合体（MHC）是由紧密连锁的高度多态的基因位点所组成的染色体上的一个遗传区域,它在动物机体的免疫系统中发挥着非常重要的作用。应用PCR-RFLP技术首次对多浪羊的MHC-DRB3的外显子2进行分子遗传多态性检测与分析。结果显示,多浪羊MHC-DRB3基因的外显子2在TaqⅠ、PstⅠ和HaeⅢ酶切位点存在多态,其酶切位点分别由2、2和6种共显性等位基因控制。综合3种酶切结果,本实验研究在多浪羊中检测到了DRB3基因的24种等位基因。 Abstract:MHC is a chromosomal region consisting of a group of closely linked loci which are highly polymorphic,and plays a central role in the immune system.The restrictive polymorphism of MHC-DRB3 exon2 in Dolang sheep was Analyzed by PCR-RFLPs.The results revealed extensive polymorphisms 2,2 and 6 RFLP types of PCR products were found with enzymes TaqⅠ,PstⅠ and HaeⅢ respectively.Considering all restrictive pattern,24 alleles for DRB3 locus were found in Dolang sheep.     

Wavelet Analysis of DNA Walks on the Human and Chimpanzee MAGE/CSAG-palindromes

The palindrome is one class of symmetrical duplications with reverse complementary characters,which is widely distributed in many organisms.Graphical representation of DNA sequence provides a simple way of viewing and comparing various genomic structures.Through 3-D DNA walk analysis,the similarity and differences in nucleotide composition,as well as the evolutionary relationship between human and chimpanzee MAGE/CSAG-palindromes,can be clearly revealed.Further wavelet analysis indicated that duplicated segments have irregular patterns compared to their surrounding sequences.However,sequence similarity analysis suggests that there is possible common ancestor between human and chimpanzee MAGE/CSAG-palindromes.Based on the specific distribution and orientation of the repeated sequences,a simple possible evolutionary model of the palindromes is suggested,which may help us to better understand the evolutionary course of the genes and the symmetrical sequences.     

Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species

The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

Molecular Evolution of VEF-Domain-Containing PcG Genes in Plants

Arabidopsis VERNALIZATION2 （VRN2）, EMBRYONIC FLOWER2 （EMF2）, and FERTILIZATION-INDEPENDENT SEED2 （FIS2） are involved in vernalization-mediated flowering, vegetative development, and seed development, respectively. Together with Arabidopsis VEF-L36, they share a VEF domain that is conserved in plants and animals. To investigate the evolution of VEF-domain-containing genes （VEF genes）, we analyzed sequences related to VEF genes across land plants. To date, 24 full-length sequences from 11 angiosperm families and 54 partial sequences from another nine families were identified. The majority of the full-length sequences identified share greatest sequence similarity with and possess the same major domain structure as Arabidopsis EMF2. EMF2-1ike sequences are not only widespread among angiosperms, but are also found in genomic sequences of gymnosperms, lycophyte, and moss. No FIS2- or VEF-L36-1ike sequences were recovered from plants other than Arabidopsis, including from rice and poplar for which whole genomes have been sequenced. Phylogenetic analysis of the full-length sequences showed a high degree of amino acid sequence conservation in EMF2 homologs of closely related taxa. VRN2 homologs are recovered as a clade nested within the larger EMF2 clade. FIS2 and VEF-L36 are recovered in the VRN2 clade. VRN2 clade may have evolved from an EMF2 duplication event that occurred in the rosids prior to the divergence of the eurosid I and eurosid II lineages. We propose that dynamic changes in genome evolution contribute to the generation of the family of VEF-domain-containing genes, Phylogenetic analysis of the VEF domain alone showed that VEF sequences continue to evolve following EM F2NRN2 divergence in accordance with species relationship. Existence of EMF2-1ike sequences in animals and across land plants suggests that a prototype form of EMF2 was present prior to the divergence of the plant and animal lineages. A proposed sequence of events, based on domain organization and occurrence of intermediate seque     

ACE基因多态性与高血压肾脏损害及PAI-1的关系

为探讨血管紧张素转换酶（ACE）基因多态性与高血压肾损害和纤溶酶原激活物抑制物-1（PAI-1）的关系,应用聚合酶链反应（PCR）检测96例正常人、67例高血压无肾脏损害患者和70例高血压伴肾损害患者的ACE基因型,采用ELISA法检测血浆PAI-1。ACE基因I/D多态性与高血压病无明显相关,但高血压肾损害患者DD基因型频率及D等位基因频率显著高于对照组和高血压无肾脏损害组,χ2值分别为6.8589、5.6162 和5.9085、5372。血浆PAI-1在DD型、ID型、II型高血压患者之间亦有显著性差异（P＜0.05）。ACE基因DD型可能是高血压肾损害的危险因素;ACE基因多态性与血浆PAI-1水平相关。 Abstract:The work is to explore the relationship between the polymorphism of angiotensin converting enzyme(ACE) gene and hypertensive kidney lesion/PAI-1 in hypertension patients.ACE genotyping with polymorase chain reaction (PCR) was performed in 96 unrelated healthy controls,67 hypertensives without kidney lesion and 70 hypertensives complicated with kidney lesion.The plasma PAI-1 were determined with ELISA.No significant differences could be detected between ACE gene I/D polymorphism and hypertension.However,the frequencies of DD genotype and deletion allele among the hypertensives complicated with kidney lesion were higher than those among the healthy controls and those among the hypertensives without kidney lesion."χ2" values were 6.8589,5.6162 and 5.9085,5.372 respectively.The plasma PAI-1 level showed significant differences among DD genotype,ID genotype and II genotype(P＜0.05).The DD genotype of ACE gene may be a risk for hypertensive kidney lesion.The plasma PAI-1 level is associated with ACE gene polymorphism.     

山东临朐人群HLA等位基因多态性 与幽门螺杆菌感染关系的研究

为了研究山东临朐地区健康人白细胞抗原（HLA）Ⅰ类等位基因多态性与幽门螺杆菌（Hp）感染的关系,运用序列特异性引物聚合酶链反应（PCR SSP）的方法,检测Hp阳性人群（90例）和Hp阴性人群（49例）的HLA Ⅰ类和Ⅱ类等位基因。结果在HLA Ⅰ类（A、B、CW）的共68个等位基因多态中,发现在感染及非感染人群中存在4个显著性差异的位点;在HLA Ⅱ类（DRB1、DQB1和DRB3、DRB4、DRB5）的共22个等位基因多态中,没有发现显著性差异的位点。A*02等位基因频率,Hp阳性低于阴性人群（OR,0.56;95%CI,0.33～0.94;P,0.029）;B*48等位基因频率,Hp阳性低于阴性人群（OR,0.15;95%CI,0.03～0.72;P,0.007）;CW*08〖STBZ〗等位基因频率,Hp阳性低于Hp阴性人群（OR,0.32;95% CI,0.15～0.69;P,0.003）;CW*15等位基因频率,Hp阳性高于Hp阴性人群（OR,5.11;95%CI,0.63～40.90;P,0.024）。结果表明HLA Ⅰ类等位基因的多态性可能与山东临朐地区Hp的易感性有关;HLA Ⅱ类等位基因的多态性可能与其无关。HLA Ⅰ类等位基因中,CW*15可能是Hp感染的易感基因;A*02、B*48和CW*08可能是保护性基因。Abstract: In order to analyze the relationship of HLA polymorphisms and the infection of H.pylori in the population of Linqu County in Shandong Province,polymerase chain reaction with sequence specific primers(PCR SSP) was used to determine the alleles of HLA typeⅠand Ⅱ in 90 Hp positive persons and 49 Hp negative controls.The results showed that among the 68 alleles of HLA typeⅠ,4 alleles were found significantly different between Hp positive and Hp negative population,while no significant difference was found among the 22 alleles of HLA typeⅡ.Hp positive persons had a lower allele frequency of A*02（OR=0.56,95% CI=0.33～0.94;P=0.029）, B*48（OR=0.15,95% CI=0.03～0.72;P=0.007）,CW*08（OR=0.32,95% CI=0.15～0.69;P=0.003）and a higher allele frequency of CW*15（OR=5.11,95% CI=0.63～40.90;P=0.024）compared with Hp negative controls.Our results indicated that the polymorphisms of HLA typeⅠis involved in the genetic susceptibility of Hp infection in Linqu County,while the polymorphisms of HLA typeⅡ may have no relationship with the genetic susceptibility of Hp infection.It was shown that among the alleles of HLA typeⅠ,CW*15might be a susceptible gene of Hp infection while A*02,B*48 and CW*08might be protective genes.     

实验用SPF大白猪和长白猪SLAⅡ类基因多态性研究

目的研究实验用SPF大白猪和长白猪SLA II类基因的多态性。方法分别采集15头SPF大白猪和22头长白猪抗凝血,分离外周血淋巴细胞,提取总RNA,反转录后RT-PCR扩增DQB1、DRB1和DQA基因并进行测序,分析获得的SLA II类等位基因序列多态性。结果 3个基因共获得25个等位基因,包括8个DQB1,10个DRB1和7个DQA,全部获得ISAG SLA命名委员会的官方命名,其中3个等位基因首次提交完整序列,命名为DQB1*02:12(KU754590)、DQB1*02:03(KU754591)和DRB1*06:07(KU754601),3个DQA等位基因为新发现等位基因。SPF大白猪和长白猪DQB1等位基因与外源性抗原结合的15个氨基酸中,共有5个氨基酸具有高度保守性;DRB1等位基因的16个外源性抗原识别位点中,仅1个位点高度保守;DQA等位基因19个抗原结合位点中,有11个高度保守。SLA II类基因氨基酸序列分子进化树表明,3个基因分别聚为两大类,与国外Yucatan小型猪具有较近的亲缘关系,而与其他猪种未表现明显遗传距离相隔。结论成功鉴定了大白猪和长白猪的25个SLA II类等位基因,发现其具有较为丰富的多态性,所获得SLA II类等位基因在其他品种猪也广泛分布,具有多样性,这一研究结果对大白猪和长白猪发展为经典实验动物模型具有重要作用。     

中国南方汉族群体MPSI型Kpn I酶切位点的遗传多态性

为研究中国汉族群体IDUA 基因Kpn I 酶切位点的遗传多态性以及该位点等位基因片段传递的规律, 采用PCR-RFLP技术, 对162例无血缘关系的健康中国汉人的324条 染色体进行检测,另又对5个家系16位成员进行同样的检测,然后用χ2检验进行统计学处理。结果表明,等位基因A1 频率为0.17,等位基因A2 频率为0.83,杂合率为29%;A1 、A2 的传递规律与理论上预计的完全符合。认为中国汉族群体IDUA 基因 Kpn I 酶切位点也具有遗传多态性, 并且与国外报道的无显著性差异;A1、A2 在世代中的传递完全符合孟德尔遗传规律。 Abstract:To investigate the genetic polymorphism of the Kpn I site in the α-L-iduronidase(IDUA) gene from a Han population in southern China and to study the mode of transmission of alleles, PCR-RFLP was used to analyze 324 chromosomes from 162 Chinese unrelated healthy Han individuals, and the analysis of the genotypes of 16 members in five families. To compare the frequencies and heterzygosity between Chinese Han population and Caucasians in Western by using χ2test. The frequency of allele 1 (450bp) was 0.17,allele 2 (390 plus 60 bp) 0.83, the heterozygosity was 29%.The genotypes of each member of all families detected was completely agreement with the theorical assessment. The locus of Kpn I in the IDUAgene from Han population has polymorphism. There is no significant difference between Chinese Han population and Caucasians in Western countries. The transmission of alleles was agreement with the Mendelian genetic law.     

Isolation and characterization of wheat ω-gliadin genes

The DNA sequences of two full-length wheat ω-gliadin prolamin genes (ωF20b and ωG3) containing significant 5′ and 3′ flanking DNA sequences are reported. The ωF20b DNA sequence contains an open reading frame encoding a 30,460-Dalton protein, whereas the ωG3 sequence would encode a putative 39,210-Dalton protein except for a stop codon at amino-acid residue position 165. These two ω-gliadin genes are closely related and are of the ARQ-/ARE-variant type as categorized by the derived N-terminal amino-acid sequences and amino-acid compositions. The ω-gliadins were believed be related to the ω-secalins of rye and the C-hordeins of barley, and analyses of these complete ω-gliadin sequences confirm this close relationship. Although the ω-type sequences from all three species are closely related, in this analysis the rye and barley ω-type sequences are the most similar in a pairwise comparison. A comparison of ω-gliadin flanking sequences with respect to that of their orthologs and with respect to wheat gliadin genes suggests the conservation of flanking DNA necessary for gene function. Sequence data for members of all major wheat prolamin families are now available. Received: 24 August 2000 / Accepted: 15 December 2000     

毛冠鹿ZFY、ZFX基因片段的克隆与性别鉴定

根据人和鼠性别分化相关的ZFY、ZFX基因序列设计引物,以雌雄毛冠鹿的基因组DNA为模板进行PCR扩增,将扩增产物克隆到pMD18T上,获得ZFY、ZFX重组克隆,并测定了ZFY、ZFX基因片段的序列,序列比较显示两者同源性达 91%,仅在少数位点有差异,以此确定AvaⅡ为ZFX上特异酶切位点,通过PCR扩增和AvaⅡ特异酶切对毛冠鹿性别进行鉴定。 Abstract: According to the human sex differentiation related ZFY and ZFX genes, a pair of primers were designed , and fragments were amplified from the genomic DNA of male or female tufted deer. Subsequently the amplified fragments were cloned into the vector pMD18T and were sequenced. It is found that the sequences of ZFY gene and ZFX gene have 91% homology. Based on the different nucleotides, restriction site of AvaⅡ was found to be specific to ZFX gene. The results show that the combination of PCR with AvaⅡ digestion is a simple and sensitive way to identify the tufted deer sex.