P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster

We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.     

Tandem array of human visual pigment genes at Xq28

Unequal crossing-over within a head-to-tail tandem array of the homologous red and green visual pigment genes has been proposed to explain the observed variation in green-pigment gene number among individuals and the prevalence of red-green fusion genes among color-blind subjects. This model was tested by probing the structure of the red and green pigment loci with long-range physical mapping techniques. The loci were found to constitute a gene array with an approximately 39-kilobase repeat length. The position of the red pigment gene at the 5' edge of the array explains its lack of variation in copy number. Restriction maps of the array in four individuals who differ in gene number are consistent with a head-to-tail configuration of the genes. These results provide physical evidence in support of the model and help to explain the high incidence of color blindness in the human population.     

The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility

Segmental duplications in the human genome are selectively enriched for genes involved in immunity, although the phenotypic consequences for host defense are unknown. We show that there are significant interindividual and interpopulation differences in the copy number of a segmental duplication encompassing the gene encoding CCL3L1 (MIP-1alphaP), a potent human immunodeficiency virus-1 (HIV-1)-suppressive chemokine and ligand for the HIV coreceptor CCR5. Possession of a CCL3L1 copy number lower than the population average is associated with markedly enhanced HIV/acquired immunodeficiency syndrome (AIDS) susceptibility. This susceptibility is even greater in individuals who also possess disease-accelerating CCR5 genotypes. This relationship between CCL3L1 dose and altered HIV/AIDS susceptibility points to a central role for CCL3L1 in HIV/AIDS pathogenesis and indicates that differences in the dose of immune response genes may constitute a genetic basis for variable responses to infectious diseases.     

Natural selection shapes genome-wide patterns of copy-number polymorphism in Drosophila melanogaster

The role that natural selection plays in governing the locations and early evolution of copy-number mutations remains largely unexplored. We used high-density full-genome tiling arrays to create a fine-scale genomic map of copy-number polymorphisms (CNPs) in Drosophila melanogaster. We inferred a total of 2658 independent CNPs, 56% of which overlap genes. These include CNPs that are likely to be under positive selection, most notably high-frequency duplications encompassing toxin-response genes. The locations and frequencies of CNPs are strongly shaped by purifying selection, with deletions under stronger purifying selection than duplications. Among duplications, those overlapping exons or introns, as well as those falling on the X chromosome, seem to be subject to stronger purifying selection.     

Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia

Schizophrenia is a devastating neurodevelopmental disorder whose genetic influences remain elusive. We hypothesize that individually rare structural variants contribute to the illness. Microdeletions and microduplications >100 kilobases were identified by microarray comparative genomic hybridization of genomic DNA from 150 individuals with schizophrenia and 268 ancestry-matched controls. All variants were validated by high-resolution platforms. Novel deletions and duplications of genes were present in 5% of controls versus 15% of cases and 20% of young-onset cases, both highly significant differences. The association was independently replicated in patients with childhood-onset schizophrenia as compared with their parents. Mutations in cases disrupted genes disproportionately from signaling networks controlling neurodevelopment, including neuregulin and glutamate pathways. These results suggest that multiple, individually rare mutations altering genes in neurodevelopmental pathways contribute to schizophrenia.     

Activated expression of the N-myc gene in human neuroblastomas and related tumors

In neuroblastoma lines in which the N-myc gene is present as a single copy, the expression of N-myc as messenger RNA is increased relative to that in nonneuroblastoma cell lines and tumors. The increase of expression in neuroblastomas with amplified N-myc genes is the result of (i) an increase in the absolute amount of expression of each N-myc gene and (ii) an increase in the copy number of the N-myc gene. A second gene--which is amplified in many of the same lines as N-myc--is expressed to about the same degree in most human cell lines and primary tumors regardless of origin (when normalized to gene copy number). Thus, a change in the regulation of N-myc expression in neuroblastomas and certain other tumors results in greatly increased expression of each N-myc gene copy.     

A gain-of-function polymorphism controlling complex traits and fitness in nature

Identification of the causal genes that control complex trait variation remains challenging, limiting our appreciation of the evolutionary processes that influence polymorphisms in nature. We cloned a quantitative trait locus that controls plant defensive chemistry, damage by insect herbivores, survival, and reproduction in the natural environments where this polymorphism evolved. These ecological effects are driven by duplications in the BCMA (branched-chain methionine allocation) loci controlling this variation and by two selectively favored amino acid changes in the glucosinolate-biosynthetic cytochrome P450 proteins that they encode. These changes cause a gain of novel enzyme function, modulated by allelic differences in catalytic rate and gene copy number. Ecological interactions in diverse environments likely contribute to the widespread polymorphism of this biochemical function.     

鸭PRLR基因拷贝数的变异

本研究以台湾褐色菜鸭为试验动物,采用荧光定量PCR技术,使用2-ΔΔCt换算法对PRLR基因拷贝数进行定量,分析了其多态性与台湾褐色菜鸭生产性能的相关性.结果表明,PRLR和Ldh-B标准曲线的R2分别为0.991和0.990,扩增效率分别为111.68%和108.429%,斜率分别为-3.070和-3.135,PRLR和Ldh-B的扩增效率近似一致,可通过2-ΔΔCt方法对PRLR基因进行定量分析.在94羽台湾褐色菜鸭的样品中共检测到5种拷贝数(1、2、3、4、5)变异类型,拷贝数变异与台湾褐色菜鸭的蛋壳厚度和蛋形指数显著相关(P0.05),拷贝数为2的个体蛋壳厚度显著高于拷贝数为1和5的个体;拷贝数为3的个体蛋形指数显著低于拷贝数为1、2和4的个体;其余性状均无显著相关(P0.05).因此,PRLR基因拷贝变异区域可能影响蛋壳厚度和蛋形指数.     

The fine-scale structure of recombination rate variation in the human genome

The nature and scale of recombination rate variation are largely unknown for most species. In humans, pedigree analysis has documented variation at the chromosomal level, and sperm studies have identified specific hotspots in which crossing-over events cluster. To address whether this picture is representative of the genome as a whole, we have developed and validated a method for estimating recombination rates from patterns of genetic variation. From extensive single-nucleotide polymorphism surveys in European and African populations, we find evidence for extreme local rate variation spanning four orders in magnitude, in which 50% of all recombination events take place in less than 10% of the sequence. We demonstrate that recombination hotspots are a ubiquitous feature of the human genome, occurring on average every 200 kilobases or less, but recombination occurs preferentially outside genes.     

Multiple rare alleles contribute to low plasma levels of HDL cholesterol

Heritable variation in complex traits is generally considered to be conferred by common DNA sequence polymorphisms. We tested whether rare DNA sequence variants collectively contribute to variation in plasma levels of high density lipoprotein cholesterol (HDL-C). We sequenced three candidate genes (ABCA1, APOA1, and LCAT) that cause Mendelian forms of low HDL-C levels in individuals from a population-based study. Nonsynonymous sequence variants were significantly more common (16% versus 2%) in individuals with low HDL-C (95th percentile). Similar findings were obtained in an independent population, and biochemical studies indicated that most sequence variants in the low HDL-C group were functionally important. Thus, rare alleles with major phenotypic effects contribute significantly to low plasma HDL-C levels in the general population.     

C-type natriuretic peptide stimulates chicken myoblast differentiation through NPRB/NPRC receptors and metabolism pathway

Skeletal muscle development is closely related with the amount of meat production and its quality in chickens. Natriuretic peptides (NPs) play an important role in myotube formation and fat oxidation of skeletal muscle in animals. The effect of C-type natriuretic peptide (CNP), an important member of the NPs, and its underlying molecular mechanisms in skeletal muscle are incompletely understood. Treatment of myoblasts with CNP led to enhanced proliferation/differentiation and significantly upregulated (P<0.05) mRNA expression of the CNP receptors natriuretic peptide receptor B (NPRB) and the clearance receptor C (NPRC). In cells exposed to CNP, 142 differentially expressed genes (84 up-regulation and 58 down-regulation) (P<0.05) were identified by RNA-sequencing compared with those in control cells. Sixteen genes were significantly enriched (P<0.05) in the metabolic pathway, and six of them (phospholipase C β4, phospholipase C β2, phosphoglycerate mutase 1, creatine kinase B, peroxiredoxin 6 and CD38) were closely related to skeletal muscle development and differentially expressed. In conclusion, CNP stimulated differentiation of myoblasts by upregulating expression of the NPRB and NPRC receptors and enriching key genes in the metabolic pathway.     

Strong association of de novo copy number mutations with autism

We tested the hypothesis that de novo copy number variation (CNV) is associated with autism spectrum disorders (ASDs). We performed comparative genomic hybridization (CGH) on the genomic DNA of patients and unaffected subjects to detect copy number variants not present in their respective parents. Candidate genomic regions were validated by higher-resolution CGH, paternity testing, cytogenetics, fluorescence in situ hybridization, and microsatellite genotyping. Confirmed de novo CNVs were significantly associated with autism (P = 0.0005). Such CNVs were identified in 12 out of 118 (10%) of patients with sporadic autism, in 2 out of 77 (3%) of patients with an affected first-degree relative, and in 2 out of 196 (1%) of controls. Most de novo CNVs were smaller than microscopic resolution. Affected genomic regions were highly heterogeneous and included mutations of single genes. These findings establish de novo germline mutation as a more significant risk factor for ASD than previously recognized.     

CYP71AV1和CPR基因在转基因青蒿后代中的遗传分析

为研究CYP71AV1和CPR基因在转基因青蒿后代中的遗传及表达特性,在获得共转CYP71AV1和CPR基因青蒿(GYR)T0代的基础上,对其T2代用普通PCR和Southern Blot分别检测了目标基因分离比及部分植株中目标基因拷贝数;利用实时定量PCR分析了部分植株中目标基因的转录水平;利用HPLC-ELSD测定了其青蒿素含量;对其关键农艺性状也进行了测定。结果表明虽然目标基因在世代间可以顺利遗传,但其拷贝数则有一些不规则的变化,转基因位点的纯合较难实现;目标基因的表达也受到基因组的修饰限制系统影响。T2代青蒿素含量最高的一组平均含量比对照提高46.9%,植株鲜重和叶片干重则没有显著差异。本研究为进一步获得遗传稳定的转基因青蒿株系打下了基础。     

基于SSR标记的中国绿豆种质资源遗传多样性研究

【目的】分析中国栽培绿豆种质资源的遗传多样性、亲缘关系和遗传分化,为资源的有效利用、新基因的挖掘和新品种选育奠定基础。【方法】利用40对SSR引物对18个不同地理来源（共272份种质）的绿豆群体进行遗传多样性分析。【结果】共检测到125个等位基因,平均等位基因数（NA）为3.1个,平均有效等位基因数（NE）为1.8个,平均Nei’s基因多样性（H）为0.4233,平均多态性信息含量(PIC)为0.3497,平均期望杂合度（He）为0.4241,平均Shannon信息指数（I）为0.6754,比较发现,河北、山东和安徽是绿豆资源遗传变异较为丰富的地区;平均观测杂合度（Ho）为0.1001,种群内总近交系数（Fis）为0.6759,表明中国绿豆种质间存在一定程度地近交现象;18个参试群体整体水平上的基因流（Nm）值为0.6936,种群间遗传分化系数（Fst）为0.2649,遗传变异水平较高;基于Popgene软件的聚类结果可将272份参试个体聚为2大类,将18个参试群体分为3大类,群体间地理来源越近,亲缘关系也越近。【结论】中国绿豆种质资源遗传多样性较高;地理生态条件等对绿豆种质资源的遗传变异影响很大;群体间遗传分化较大,但同时也存在一定程度地近交现象。     

Tandem duplication of D-loop and ribosomal RNA sequences in lizard mitochondrial DNA

Some Cnemidophorus exsanguis have mitochondrial DNA's (mtDNA's) that are 22.2 kilobases (kb) in size, whereas most have mtDNA's of 17.4 kb. Restriction site mapping, DNA transfer hybridization experiments, and electron microscopy show that the size increment stems from the tandem duplication of a 4.8-kb region that includes regulatory sequences and transfer and ribosomal RNA genes. This observation is notable in that sequences outside of the control region are involved in major length variation. Besides revealing a novel form of mtDNA evolution in animals, these duplications provide a useful system for investigating the molecular and evolutionary biology of animal mtDNA.