抗体选择压作用下H9N2亚型禽流感病毒NA基因的变异

将LG1株H9N2亚型禽流感病毒在带有抗LG1株母源抗体的鸡胚中分4个独立系列连续传40代后,有3个系列从10～20代起在NA基因的#99位发生了可稳定遗传的碱基"G"到"A"的突变,并使氨基酸由蛋氨酸变为异亮氨酸;有2个系列从20～30代起在#473位发生了可稳定遗传的由"A"到"G"的碱基突变,导致相应的氨基酸由天冬酰胺变为丝氨酸,另一个传代系列在50代时也发生了同样的突变。在无抗体的鸡胚上的2个独立对照系列同样传了80代,在这2个位点没有发生突变,表明这2个突变与抗体的选择压相关。在抗LG1母源抗体阳性鸡胚的连续40代传代过程中,NA基因在有抗体组的四个传代系列碱基的非同义突变(NS)与同义突变(S)比为4.6(32/7),而在无抗体组NS/S比为2.0(16/8)。有抗体组NS/S值显著高于无抗体组,也显示出抗体的选择压作用。     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%–99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%–96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110–120, aa#141–151 and aa#189–194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

<Emphasis Type="Italic">RET</Emphasis> gene mutations and polymorphisms in medullary thyroid carcinomas in Indian patients

Germline mutations of RET gene are pathognomonic of multiple endocrine neoplasia (MEN; MEN 2A/MEN 2B) and familial medullary thyroid carcinoma (FMTC), constituting 25% of medullary thyroid carcinomas (MTCs). We investigated RET gene mutations and polymorphisms at exons 10, 11, 13, 14, 15 and 16 in 140 samples, comprising 51 clinically diagnosed MTC patients, 39 family members of patients and 50 normal individuals. The method of choice was PCR and direct nucleotide sequencing of the PCR products. RET gene mutations were detected in 15 (29.4%) patients, with MEN 2A/FMTC in 13 patients and MEN 2B in 2 patients. Further, 39 family members of seven index cases were analysed, wherein four of the seven index cases showed identical mutations, in 13 of 25 family members. We also examined single nucleotide polymorphisms (SNPs) in RET gene exons in 101 unrelated samples. Significant differences in the allelic frequencies of SNPs at codons 691, 769, 836 and 904 between patient and control groups were not observed. However, SNP frequencies were significantly different in the Indian group as compared with other European groups. We identified two novel, rare and unique SNPs separately in single patients. Our study demonstrated presence of MEN 2A/MEN 2B/FMTC-associated mutations in accordance with the reported literature. Thus, RET gene mutations in exons 10, 11, 13, 14, 15 and 16 constitute a rapid test to confirm diagnosis and assess risk of the disease in familial MEN 2A/MEN 2B/FMTC.     

Mutations in the <Emphasis Type="Italic">S</Emphasis> gene region of hepatitis B virus genotype <Emphasis Type="Italic">D</Emphasis> in Turkish patients

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the ‘a’-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M → T) and 127 (T → P) were found on the first loop of ‘a’-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.     

抗体选择压作用下新城疫病毒HN和F基因的演化及其抗原性变异的比较分析

TZ060107株新城疫病毒(NDV)在含有对它抗体的鸡胚成纤维细胞(CEF)培养上分3个独立系列连传50代,每10代扩增其HN和F基因并测序。选择变异最大的系列A1-50病毒,再在含有抗A1-50抗体的CEF培养上分3个独立系列连续传50代,同时设3个不带抗体的独立传代系列作为对照。对第60、70、80、90、100代病毒的HN和F基因序列比较结果显示,有抗体组HN基因的非同义突变(NS)对同义突变(S)比值NS/S为5.25,明显高于无抗体组NS/S的2.375。前50代在抗体选择压作用下已发生的稳定NS突变在含有抗A1-50抗体的细胞培养中传代仍能稳定保持,且又出现了一个新的稳定的NS突变位点。在有抗体组经传50代后F基因发生的稳定非同义突变,在抗A1-50血清作用下再连传50代后也仍然保持,且又出现3个新的稳定的NS突变。不同传代病毒与原始病毒间的血清交叉血球凝聚抑制试验结果表明,随着在含有抗NDV血清的细胞培养上传代代数的增加,病毒与原始病毒间在抗原性的差异越来越大。     

红莲型细胞质雄性不育水稻线粒体atp6基因转录本的编辑位点研究

以红莲（HL）型水稻细胞质雄性不育系A、保持系B及杂种一代F₁为材料,首次比较研究了红莲型水稻线粒体atp6基因转录本的编辑位点及各位点的编辑频率.结果表明atp6基因的转录本有18个编辑位点,其中有15个发生在密码子的第一和第二位点上,这些位点的编辑最终会导致氨基酸种类的变化.18个编辑位点在A、B和F₁中没有差异,但各位点的编辑频率在引入了核恢复基因的条件下发生了较大的变化,完全编辑的比例增加.这些结果首次证明HL型细胞质雄性不育与线粒体atp6转录本的编辑有一定相关性,编辑不充分的转录产物最终会干扰线粒体功能的正常发挥.     

Inheritance of seed coat color genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) and tagging the genes using SRAP,SCAR and SNP molecular markers

Seed coat color inheritance in Brassica napus was studied in F₁, F₂, F₃ and backcross progenies from crosses of five black seeded varieties/lines to three pure breeding yellow seeded lines. Maternal inheritance was observed for seed coat color in B. napus, but a pollen effect was also found when yellow seeded lines were used as the female parent. Seed coat color segregated from black to dark brown, light brown, dark yellow, light yellow, and yellow. Seed coat color was found to be controlled by three genes, the first two genes were responsible for black/brown seed coat color and the third gene was responsible for dark/light yellow seed coat color in B. napus. All three seed coat color alleles were dominant over yellow color alleles at all three loci. Sequence related amplified polymorphism (SRAP) was used for the development of molecular markers co-segregating with the seed coat color genes. A SRAP marker (SA12BG18388) tightly linked to one of the black/brown seed coat color genes was identified in the F₂ and backcross populations. This marker was found to be anchored on linkage group A9/N9 of the A-genome of B. napus. This SRAP marker was converted into sequence-characterized amplification region (SCAR) markers using chromosome-walking technology. A second SRAP marker (SA7BG29245), very close to another black/brown seed coat color gene, was identified from a high density genetic map developed in our laboratory using primer walking from an anchoring marker. The marker was located on linkage group C3/N13 of the C-genome of B. napus. This marker also co-segregated with the black/brown seed coat color gene in B. rapa. Based on the sequence information of the flanking sequences, 24 single nucleotide polymorphisms (SNPs) were identified between the yellow seeded and black/brown seeded lines. SNP detection and genotyping clearly differentiated the black/brown seeded plants from dark/light/yellow-seeded plants and also differentiated between homozygous (Y2Y2) and heterozygous (Y2y2) black/brown seeded plants. A total of 768 SRAP primer pair combinations were screened in dark/light yellow seed coat color plants and a close marker (DC1GA27197) linked to the dark/light yellow seed coat color gene was developed. These three markers linked to the three different yellow seed coat color genes in B. napus can be used to screen for yellow seeded lines in canola/rapeseed breeding programs.     

家蚕二分浓核病毒ns1基因序列的改造、表达和鉴定

探讨翻译起始区(TIR)部分密码子发生同义突变后,对家蚕二分浓核病毒(BmBDV)ns1基因表达的影响,以及对BmBDV NS1蛋白毒性进行鉴定,设计特异性上游引物,对BmBDV ns1基因中第3、4、9和10个密码子进行同义突变,利用原核表达系统对野生型和改造后的ns1序列进行表达,通过SDS-PAGE电泳对这两种序列的表达产量进行分析。利用Protein Iso~(TM)GST Resin从超声破碎的菌液上清中纯化融合有GST的NS1蛋白,进而对纯化的靶蛋白在细胞水平和家蚕体内进行毒性分析。结果表明:TIR突变后的BmBDV ns1序列,其与野生型序列的表达产量之间没有明显差异;BmBDV NS1蛋白具有抑制细胞增殖和诱导家蚕致死的生化活性。     

Molecular mapping of leaf rust resistance gene <Emphasis Type="Italic">LrZH84</Emphasis> in Chinese wheat line Zhou 8425B

Leaf rust, caused by Puccinia triticina, is one of the most widespread diseases in common wheat (Triticum aestivum L.) worldwide. With the objective of identifying and mapping new genes for resistance to leaf rust, F₁, F₂ plants and F₃ lines from a cross between resistant line Zhou 8425B and susceptible line Chinese Spring were inoculated with Chinese P. triticina races THTT and MBHP in the greenhouse. A total of 793 pairs of SSR primers were used to test the parents and resistant and susceptible bulks. Seven polymorphic chromosome 1B markers were used for genotyping the F₂ and F₃ populations. Zhou 8425B carried a single dominant resistance gene, temporarily designated LrZH84, linked to SSR markers gwm582 and barc8 with genetic distances of 3.9 and 5.2 cM, respectively. The Xbarc8 allele co-segregated with Lr26 in the F₃ population. The Xgwm582 allele associated with LrZH84 was identified as a leaf rust resistance gene and shown to be present in the Predgornaia 2 parent of Zhou 8425B. The seedling reaction pattern of LrZH84 was different from those of lines with Lr26, Lr33, Lr44 and Lr46, all of which are located in chromosome 1B. It was concluded that LrZH84 is likely to be a new leaf rust resistance gene.     

抗体选择压作用下H9N2亚型禽流感病毒HA基因的变异

摘要：【目的】了解H9N2亚型禽流感病毒(AIV)在抗体选择压作用下的遗传变异。【方法】将制备疫苗用的LG1株H9N2亚型AIV分别接种含有母源抗体鸡胚(A组)和不含有母源抗体的SPF鸡胚(B组),并连续传代。其中A组再分为4 个独立的传代系列A1-4,B组再分为2 个独立传代系列B1-2。在每个传代系列,分别对第10,20,30,40,50 代病毒的HA基因进行扩增克隆测序,并与原始病毒的序列比较。【结果】LG1株H9N2在没有抗体的鸡胚的传代过程中,仅发生少数碱基的不稳定随机变异,且多为无义突变。在2 个传代系列的10 个代次病毒,共出现了29 个位点变异,有义突变(NS)与无义突变(S)比值NS/S为1.42 。但在有抗体的鸡胚的传代过程中,发生了多个呈现稳定遗传的有义突变。在4 个传代系列的20 个代次病毒,共出现了45 个位点变异,有义突变(NS)与无义突变(S)比值NS/S为3.46。【结论】在鸡胚传代过程中母源抗体提供的免疫选择压确实能影响H9N2的HA基因的变异。同时表明,带有母源抗体的鸡胚是实验室条件下研究免疫选择压对病毒抗原性变异影响的一种有效的实验模型。     

A genetic linkage map of <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. and localization of genes for specific resistance to six races of anthracnose (<Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>)

A genetic map of common bean was constructed using 197 markers including 152 RAPDs, 32 RFLPs, 12 SCARs, and 1 morphological marker. The map was established by using a F₂ population of 85 individuals from the cross between a line derived from the Spanish landrace Andecha (Andean origin) and the Mesoamerican genotype A252. The resulting map covers about 1,401.9 cM, with an average marker distance of 7.1 cM and includes molecular markers linked to disease resistance genes for anthracnose, bean common mosaic virus, bean golden yellow mosaic virus, common bacterial blight, and rust. Resistance to races 6, 31, 38, 39, 65, and 357 of the pathogenic fungus Colletotrichum lindemuthianum (anthracnose) was evaluated in F₃ families derived from the corresponding F₂ individuals. The intermediate resistance to race 65 proceeding from Andecha can be explained by a single dominant gene located on linkage group B1, corresponding to the Co-1 gene. The recombination between the resistance specificities proceeding from A252 agrees with the assumption that total resistance to races 6, 31, 38, 39, 65, and 357, is organized in two clusters. One cluster, located on B4 linkage group, includes individual genes for specific resistance to races 6, 38, 39, and 357. The second cluster is located on linkage group B11 and includes individual genes for specific resistance to races 6, 31, 38, 39, and 65. These two clusters correspond to genes Co-3/Co-9 and Co-2, respectively. It is concluded that most anthracnose resistance Co- genes, previously described as single major genes conferring resistance to several races, could be organized as clusters of different genes conferring race-specific resistance. C. Rodríguez-Suárez and B. Méndez-Vigo equally share for authorship.     

Genetic variants of <Emphasis Type="Italic">CYP1B1</Emphasis> and <Emphasis Type="Italic">WDR36</Emphasis> in the patients with primary congenital glaucoma and primary open angle glaucoma from saint-Petersburg

In 32 unrelated patients with primary congenital glaucoma (PCG), a search for mutations in the myocilin (MYOC), cytochrome P450B1 (CYP1B1), and WDR36 genes was performed. The Q368X mutation in myocilin gene, typical of the patients with adult-onset primary open-angle glaucoma (POAG), was not detected in the PCG patients. Screening of the CYP1B1 introns 2 and 3 for the presence of mutations in PCG patients revealed only six DNA polymorphisms, including IVS1-12ntT>C (g.3793 T>C), A119S (g.4160 G>T; GCC>TCC), G188G (g.4369 C>A; GGC>GGA), L432V (g.8131 C>G; CTG>GTG), D449D (g.8184 C>T; GAC>GAT), and N453S (g.8195 A>G; AAC>AGC) (nucleotide numbering is given in accordance with the GenBank sequence U56438). In the groups of PCG patients and donors without eye diseases, the frequencies of these variants were not statistically significantly different, pointing to the neutrality of these polymorphisms. Furthermore, the CYP1B1 polymorphism L432V, considered to be associated with POAG in some world populations, was not associated with this disease in the patients from St. Petersburg. DNA collections obtained from the POAG and PCG patients and from the control group were tested for the carriage of the worldwide distributed mutations of the WRD36 gene, D658G, R529Q, A449T, and N355S. D658G variant was found with equally low frequencies in the groups of POAG and PCG patients, as well as in the control group. Mutations A449T and R529Q were found only once each, while mutation N355S was not detected in any of the groups examined. Our results indicate that the WDR36 variants make no substantial contribution to the development of POAG and PCG in the patients from St. Petersburg and represent normal DNA polymorphism. It is likely that in most of the PCG patients from the population examined the disease is not associated with the CYP1B1 gene defects.     

An Uniquely Purified HCV NS3 Protease and NS4A21–34 Peptide Form a Highly Active Serine Protease Complex in Peptide Hydrolysis

The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4°C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys–Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (k_cat/K_M) of 205 and 196,000 M⁻¹ s⁻¹, respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease–NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (k_cat/K_M of 5180 ± 670 M⁻¹ s⁻¹). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed.     

Genetics and molecular mapping of resistance to necrosis inducing race 5 of <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> in tetraploid wheat

Race 5 of Pyrenophora tritici-repentis, causal agent of tan spot, induces two distinct symptoms, necrosis and chlorosis in susceptible tetraploid and hexaploid wheat, respectively. This study was conducted under controlled environmental conditions to determine the inheritance of resistance to P. tritici-repentis, race 5, in a tetraploid wheat population and to map the resistance genes. Additionally, the relationship between the resistance genes effective against necrosis inducing races 3 and 5 in tetraploid wheat was determined. A population of 98 recombinant-inbred lines (RIL) was developed from a cross between the resistant genotype Triticum turgidum # 283 (PI352519) and the susceptible durum cultivar Coulter. This RIL population was screened individually with race 3 and race 5 and molecular mapping of the resistance gene(s) in this population was conducted. Additionally, the F₂ and F_4:5 generations of this population were screened with race 5 to determine the genetic control of resistance. Plants were inoculated at the two-leaf stage and disease reaction was assessed based on 1 to 5 lesion-type rating scale eight days after inoculation. Segregation analysis of the F₂ generation and of the F_4:5 and F_6:7 families indicated that a single recessive gene controlled resistance to necrosis induced by race 5. Analysis of the mapping data of the T. turgidum # 283/Coulter RIL population indicate that a major gene, designated tsn5, controlling resistance to race 5 is located on the long arm of chromosome 3B. The tsn5 gene is 8.3 cM proximal to the gene tsn2 that controls resistance to necrosis induced by race 3.     

Evolution of gp85 gene of subgroup J avian leukosis virus under the selective pressure of antibodies

Subgroup J Avian leucosis virus (ALV-J) strain NX0101 was inoculated into chicken embryo fibroblasts (CEF) monolayers in 6-well plates. The six wells of CEF inoculated with NX0101 were divided into groups A (without anti-ALV-J serum in the medium) and B (with anti-ALV-J serum in the medium), then viruses from each well of both groups were separately passed in CEF every 6 d and formed their independent passage lineages. For each lineage of both groups, gp85 genes of the viruses in the 10th, 20th and 30th passages were amplified, cloned and sequenced. The sequence data indicated that the homologies of gp85 at aa level between the primary virus and the passed viruses of different passages of 3 lineages in group A were 97.7%-99.7%; and the homologies of gp85 between the primary virus and the passed viruses of different passages of 3 lineages in group B were 93.8%-96.1%. Analysis of the ratios of nonsynonium (NS) vs synonium (S) mutations of nucleic acids demonstrated that NS/S in 3 highly variable (hr-) regions at aa#110-120, aa#141-151 and aa#189-194 of gp85 in 3 lineages of group A were 2 (8/4), 1(3/3) and 1.3 (4/3), however, NS/S in the same 3 hr-regions of group B were 4.1 (13/3), 4.7 (14/3) and 3.3 (11/3). This study is the first demonstration of influence of immune selective pressure on evolution of ALV-J gp85 by specific antibodies under the controlled in vitro experiments.     

Genetic mapping of a new semi-dwarf gene, <Emphasis Type="Italic">sd-t(t)</Emphasis>, in indica rice and estimating of the physical distance of the mapping region

Application and functional study of dwarf and semi-dwarf genes are of great importance to both crop breeding and molecular biology. A new semi-dwarf gene, sd-t(t), non-allelic to sd-1, had been identified in an indica rice variety, Aitaiyin 2. In this study the gene was genetically mapped by using an F₂ population, which consisted of 474 individuals developed from a cross between Aitaiyin 2 and B30. The sd-t(t) gene was located between the RFLP markers R514 and R1408B with a distance of 1.1 cM to R514, and 4.5 cM to R1408B on chromosome 4. A physical contig covering the sd-t(t) mapping region was further constructed by screening a BAC library with R514 and R1408B as probes, and the physical distance between R514 and R1408B was estimated at approximately 147 kb. This result will facilitate map-based cloning of the sd-t(t) gene.     

Analysis of the coding regions of <Emphasis Type="Italic">VEGFR3</Emphasis> and <Emphasis Type="Italic">VEGFC</Emphasis> in Milroy disease and other primary lymphoedemas

Milroy disease (hereditary lymphoedema type I, MIM 153100) is a congenital onset primary lymphoedema with autosomal dominant inheritance. Mutations in the gene, vascular endothelial growth factor receptor 3, VEGFR3 (FLT4), are known to cause Milroy disease, but there is uncertainty about the prevalence of VEGFR3 mutations in patients with primary lymphoedema and more specifically in those with a phenotype that resembles Milroy disease. This study aims to address this issue and thereby delineate the Milroy disease phenotype. Fifty-two patients with primary lymphoedema were analysed for mutations in the coding regions of VEGFR3. Patients were divided into four groups: Typical Milroy disease with family history (group I), typical Milroy disease with no family history (group II), atypical Milroy disease (group III), and complex primary lymphoedema (group IV). Results demonstrated that with rigorous phenotyping the likelihood of detecting VEGFR3 mutations is optimised. Mutation prevalence is 75% in typical Milroy patients with a family history (group I) and 68% if positive family history is not a diagnostic criterion. A positive family history is not essential in Milroy disease. The likelihood of detecting VEGFR3 mutations in patients who have a phenotype which is not typical of Milroy disease is very small (<5%). For the 22 mutation positive patients, 14 novel VEGFR3 mutations were identified, two of which were in exon 22 and one in exon 17, confirming that these exons should be included in VEGFR3 analysis. No mutations were found outside the kinase domains, showing that analysis of this part of the gene is not useful for Milroy disease patients. VEGFC, which encodes the ligand for VEGFR3, was sequenced in all patients with typical Milroy disease (groups I and II) and no mutations were identified. F. C. Connell and P. Ostergaard contributed equally to this work. An erratum to this article can be found at