Description of Echinococcus granulosus genotypes in human hydatidosis in a region of southern Chile

INTRODUCTION: Echinococcus granulosus species has a wide variety in both geography and hosts; indeed, 10 genotypes have been reported in studies on material of animal origin. The aim of this study was to genotype E. granulosus obtained from human hydatid cysts. MATERIALS AND METHODS: The hydatid fluid and sand was collected from patients who underwent surgery for hepatic and pulmonary hydatidosis at Hospital Regional in Temuco, Chile, between 2004 and 2005. Two PCR systems were used: PCR Eg 9 and PCR Eg 16. The RsaI enzyme was used for RFLP. The genotype was confirmed using the sequence of one fragment of 366 bp from a mitochondrial gene (cox1). RESULTS: The DNA of protoscolices from 24 samples was analyzed, 4 of them from pulmonary cysts and 20 from hepatic cysts. The 366 bp fragment was amplified in 20 out of 24 samples (83.3%). Enzymatic digestion revealed the presence of 3 possible genotypes: in 20 out of 21 samples (95,2%), a restriction was observed corresponding to the G1 or G7 genotypes; in the remaining sample genotype G4 or G7 was observed. Sequencing confirmed the presence of G1 genotype for 19 samples and G6 genotype for the remaining sample (G4 or G7 according to PCR-RFLP). CONCLUSION: The PCR-RFLP technique enabled three possible genotypes present (G1 or G7, G4 or G7) to be established. Sequencing allowed us to decisively identify the G1 and G6 genotypes in our study group. Previous studies agree with the identification of the G1 genotype in our country. We consider it significant that the G6 genotype is present in Chile for its epidemiological implications.     

Confirmation of maternal transmission ratio distortion at Om and direct evidence that the maternal and paternal “DDK syndrome” genes are linked

The polar, preimplantation-embryo lethal phenotype known as the ``DDK syndrome' in the mouse is the result of the complex interaction of genetic factors and a parental-origin effect. We previously observed a modest degree of transmission-ratio distortion in favor of the inheritance of DDK alleles in the Ovum mutant (Om) region of Chromosome (Chr) 11, among offspring of reciprocal F<sub>1</sub>-hybrid females and C57BL/6 males. In this study, we confirm that a significant excess of offspring inherit DDK alleles from F<sub>1</sub> mothers and demonstrate that the preference for the inheritance of DDK alleles is not a specific bias against the C57BL/6 allele or a simple preference for offspring that are heterozygous at Om. Because none of the previous genetic models for the inheritance of the ``DDK syndrome' predicted transmission-ratio distortion through F₁ females, we reconsidered the possibility that the genes encoding the maternal and paternal components of this phenotype were not linked. We have examined the fertility phenotype of N₂ females and demonstrate that the inter-strain fertility of these females is correlated with their genotype in the Om region. This result establishes, directly, that the genes encoding the maternal and paternal components of the DDK syndrome are genetically linked. Received: 1 February 1997 / Accepted: 26 April 1997     

Arylamine N-acetyltransferase (NAT2) mutations and their allelic linkage in unrelated Caucasian individuals: correlation with phenotypic activity.

The polymorphic arylamine N-acetyltransferase (NAT2; EC 2.3.1.5) is supposed to be a susceptibility factor for several drug side effects and certain malignancies. A group of 844 unrelated German subjects was genotyped for their acetylation type, and 563 of them were also phenotyped. Seven mutations of the NAT2 gene were evaluated by allele-specific PCR (mutation 341C to T) and PCR-RFLP for mutations at nt positions 191, 282, 481, 590, 803, and 857. From the mutation pattern eight different alleles, including the wild type coding for rapid acetylation and seven alleles coding for slow phenotype, were determined. Four hundred ninety-seven subjects had a genotype of slow acetylation (58.9%; 95% confidence limits 55.5%-62.2%). Phenotypic acetylation capacity was expressed as the ratio of 5-acetylamino-6-formylamino-3-methyluracil and 1-methylxanthine in urine after caffeine intake. Some 6.7% of the cases deviated in genotype and phenotype, but sequencing DNA of these probands revealed no new mutations. Furthermore, linkage pattern of the mutations was always confirmed, as tested in 533 subjects. In vivo acetylation capacity of homozygous wild-type subjects (NAT2*4/*4) was significantly higher than in heterozygous genotypes (P = .001). All mutant alleles showed low in vivo acetylation capacities, including the previously not-yet-defined alleles *5A, *5C, and *13. Moreover, distinct slow genotypes differed significantly among each other, as reflected in lower acetylation capacity of *6A, *7B, and *13 alleles than the group of *5 alleles. The study demonstrated differential phenotypic activity of various NAT2 genes and gives a solid basis for clinical and molecular-epidemiological investigations.     

Maternal transmission ratio distortion at the mouse Om locus results from meiotic drive at the second meiotic division

We have observed maternal transmission ratio distortion (TRD) in favor of DDK alleles at the Ovum mutant (Om) locus on mouse chromosome 11 among the offspring of (C57BL/6 x DDK) F(1) females and C57BL/6 males. Although significant lethality occurs in this backcross ( approximately 50%), differences in the level of TRD found in recombinant vs. nonrecombinant chromosomes among offspring argue that TRD is due to nonrandom segregation of chromatids at the second meiotic division, i.e., true meiotic drive. We tested this hypothesis directly, by determining the centromere and Om genotypes of individual chromatids in zygote stage embryos. We found similar levels of TRD in favor of DDK alleles at Om in the female pronucleus and TRD in favor of C57BL/6 alleles at Om in the second polar body. In those embryos for which complete dyads have been reconstructed, TRD was present only in those inheriting heteromorphic dyads. These results demonstrate that meiotic drive occurs at MII and that preferential death of one genotypic class of embryo does not play a large role in the TRD.     

Genotyping the bovine kappa-casein locus using polymerase chain reaction]

Polymerase chain reaction (PCR) was used for detecting kappa-casein (kappa-casein) genotype in the synthetic breed (Jersey x Black and White x Holstein-Friesian) dairy cattle. The amplified 228 bp fragment includes a region, where relevant mutations lead to both the appearance of different kappa-casein alleles associated with amino acid substitutions and the appearance of new TaqI and HindIII restriction sites in ae-casein B gene. The specificity of the kappa-casein gene fragment amplification was supported by restriction analysis and Southern blot hybridization. Digestion of amplified fragment with endonucleases PstI, HindIII and/or TaqI allows detection of AA-, AB- and BB genotypes of kappa-casein. A total of 32 animals with known (18 samples) and unknown (14 samples) kappa-casein phenotypes were tested using PCR and blot hybridization. In all known cases the detected genotype confirmed the phenotype. Frequencies of the B allele and of the AB genotype in the breeding population are rather high (53.1 +/- 8.8 and 43.7%, respectively). The possibility of effective use of the PCR analysis for genotyping kappa-casein locus in bulls and their offspring has been shown. The advantages of the PCR method in large breeding programs and linkage analysis have been discussed.     

RS46(DXS548) genotyping of reproductive cells: approaching preimplantation testing of the fragile-X syndrome

In order to approach preimplantation testing for the fragile-X syndrome, we used genotyping of the polymorphic RS46(DXS548) locus closely linked to the FMR1 gene, in single reproductive cells of females. The RS46(DXS548) amplification was adjusted to the single cell level by a two-round polymerase chain reaction (PCR) procedure. Unfertilized oocytes and extruded polar bodies were subjected to PCR. RS46(DXS548) genotyping at the single cell level was successful in 95% of the samples. In two-third of the metaphase II oocytes and first polar bodies obtained from women who were heterozygous at the RS46(DXS548) locus, both maternal RS46(DXS548) alleles were observed because of crossing over during the first meiotic division. This makes gamete selection by first polar body analysis inefficient. From the allele frequencies found in 56 unrelated individuals, a heterozygote frequency of 51% was estimated, whereas the observed heterozygote frequency was 56%. The whole PCR procedure can be performed within 16 h after blastomere biopsy. Consequently, the selection and transfer of the diagnosed embryos can be carried out within an acceptable time. Therefore, preimplantation testing for the fragile-X syndrome with the RS46(DXS548) AC-repeat may be an alternative choice for prenatal testing for those carrier females who are heterozygous (informative) at the RS46(DXS548) locus.     

中国新疆维吾尔族DXS101遗传多态性研究

目的:研究DXS101位点在中国新疆维吾尔族群体中的遗传结构分布特征。方法:采用PCR扩增,变性聚丙烯酰胺凝胶电泳结合银染显带技术,检测100名(女42,男58)维吾尔族无关个体DXS101位点等位基因及基因型频率分布。结果:在女性样本中,DXS101位点检出9种等位基因和17种基因型;在男性样本中,DXS101位点检出7种等位基因。该位点在女性中的个体识别率为0.8937,多态信息量为0.8072,杂合度为0.8156,在男性中的个体识别率为0.6674。结论:群体遗传多态性指标显示DXS101位点在新疆维吾尔族群体中具有较高多态性,在维吾尔族群体法医学个体识别、亲权鉴定及群体遗传学研究中有重要应用价值。     

Genotyping and detection of multiple infections of Toxoplasma gondii using Pyrosequencing

A Pyrosequencing assay, based on SAG2 gene polymorphisms, was designed for genotyping and detection of multiple infections of Toxoplasma gondii. The assay was tested on samples spiked with DNA from single and multiple genotypes of T. gondii and also on a DNA sample from the brain of a rat with multiple infections. To evaluate the comparative efficacy of the assay, identical samples were also analysed by PCR-restriction fragment length polymorphism (RFLP) and dideoxy sequencing. The Pyrosequencing assay was found to be superior to the two conventional techniques. Genotyping and detection of multiple alleles were possible after a single PCR assay in duplex format, from both the spiked and direct samples. The simplex PCR assay enabled accurate quantification of the different alleles in the mix. In comparison, PCR-RFLP and dideoxy sequencing were neither able to unequivocally detect multiple genotype infections, nor quantify the relative concentrations of the alleles. We conclude that Pyrosequencing offers a simple, rapid and efficient means for diagnosis and genotyping of T. gondii, as well as detection and quantification of multiple genotype infections of T. gondii.     

Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR

A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.     

Registration of S alleles in Brassica campestris L by the restriction fragment sizes of SLGs

Polymorphism of SLG (the S-locus glycoprotein gene) in Brassica campestris was analyzed by PCR-RFLP using SLG-specific primers. Nucleotide sequences of PCR products from 15 S genotypes were determined in order to characterise the exact DNA fragment sizes detected in the PCR-RFLP analysis. Forty-seven lines homozygous for 27 S-alleles were used as plant material. One combination of primers, PS5 + PS 15, which had a nucleotide sequence specific to a class-I SLG, gave amplification of a single DNA fragment of approximately 1.3kb from the genomic DNA of 15 S genotypes. All the DNA fragments showed different electrophroetic profiles from each other after digestion with MboI or MspI. Different lines having the same S genotype had an identical electrophoretic profile even between the lines collected in Turkey and in Japan. Another class-I SLG-specific primer, PS 18, gave amplification of a 1.3-kb DNA fragment from three other S genotypes in combination with PS 15, and the PCR product also showed polymorphism after cleavage with the restriction endonucleases. Genetic analysis, Southern-hybridization analysis, and determination of the nucleotide sequences of the PCR products suggested that the DNA fragments amplified with these combinations of primers are class-I SLGs. Expected DNA fragment sizes in the present PCR-RFLP condition were calculated from the determined nucleotide sequence of SLG PCR products. A single DNA fragment was also amplified from six S genotypes by PCR with a combination of primers, PS3 + PS21, having a nucleotide sequence specific to a class-II SLG. The amplified DNA showed polymorphisnm after cleavage with restriction endonucleases. The cleaved fragments were detected by Southern-hybridization analysis using a probe of S ⁵ SLG cDNA, a class-IISLG. Partial sequencing revealed a marked similarity of these amplified DNA fragments to a class-II SLG, demonstrating the presence of class-I and class-II S alleles also in B. campestris. The high SLG polymorphism detected by the present investigation suggests the usefulness of the PCR-RFLP method for the identification of S alleles in breeding lines and for listing S alleles in B. campestris.     

Dominant distribution of mitochondrial DNA from recipient oocytes in bovine embryos and offspring after nuclear transfer

In the process of nuclear transfer, heteroplasmic sources of mitochondrial DNA from a donor cell and a recipient oocyte are mixed in the cytoplasm of the reconstituted embryo. The distribution of mitochondrial DNA heteroplasmy in nuclear transfer bovine embryos and resultant offspring was investigated by measuring polymorphism in the displacement loop region of mitochondrial DNA using PCR-mediated single-strand conformation polymorphism. Most offspring (20 of 21 calves) from recipient oocytes of undefined mitochondrial DNA genotypes showed different genotypes from the mitochondrial DNA of donor cells. The single calf that was an exception showed heteroplasmy, including the donor mitochondrial DNA genotype. Six cloned calves were produced from oocytes of a defined mitochondrial DNA genotype. All of these clonal members and various tissues showed only the mitochondrial DNA genotype derived from the oocyte. The mitochondrial DNA from donor cells appeared to be eliminated during early embryonic development; it gradually decreased at the early cleavage stages and was hardly detectable by the blastocyst stage. These results indicate that the genotype of mitochondrial DNA from recipient oocytes may become the dominant category of mitochondrial DNA in calves resulting from nuclear transfer.     

山西马身猪及其杂交后代H-FABP基因的PCR-RFLP检测

运用PCR-RFLP法检测马身猪及其杂交后代H-FABP基因多态性。结果表明,马身猪在Hinf Ⅰ-RFLP位点上有HH和Hh两种基因型,在HaeⅢ-RFLP位点上只有DD基因型,在HinfⅠ*-RFLP位点上有BB和Bb两种基因型,等位基因以h、D、B占优势,其频率分别为0．958、1．000和0．968。其杂交后代在HaeⅢ-RFLP位点上只检测到两种基因型DD和Dd。     

Relationship between sex ratio and time of insemination according to both time of ovulation and maturational state of oocyte

The aim of this study was to explore how some reproductive methodologies may affect the sex ratio. We first confirmed the association between the maturation stage of bovine oocytes at the time of in vitro fertilisation (IVF) and the sex ratio of in vitro-derived embryos. Secondly, we studied whether the time of insemination, prior to or after ovulation, could alter the sex ratio in sheep. In the first experiment, bovine oocytes were matured in vitro for 16 h; then oocytes were either fertilised in vitro immediately after extrusion of the first polar body or IVF was delayed for 8 h. The proportion of cleaving embryos and their development to the 8-cell stage was enhanced with delayed insemination. Moreover, delaying IVF produced a male-to-female sex ratio of 1.67:1.00, which was significantly different from the expected 1:1 ratio (p < 0.05), whereas more female embryos were produced when oocytes were fertilised in vitro immediately after polar body extrusion (sex ratio of 1.00:0.67; p < 0.05). In the second experiment, 380 ewes were inseminated at different times before or after ovulation, producing 537 lambs. Significant differences in the sex ratio were obtained when we compared the sex of the offspring of ewes inseminated during the 5 h preceding ovulation (more females) with those inseminated during the 5 h after ovulation (more males). Our results suggest that the differential ability of X- or Y-bearing spermatozoa to fertilise oocytes depending either on time of insemination or oocyte maturation state, may be due, at least partially, to 'intrinsic' differences in the physiological activity of X- or Y-bearing spermatozoa before fertilisation.     

Factors affecting the efficiency of embryo cryopreservation and rederivation of rat and mouse models

The efficiency of embryo banking for rat and mouse models of human disease and normal biological processes depends on the ease of obtaining embryos. Authors report on the effect of genotype on embryo production and rederivation. In an effort to establish banks of cryopreserved embryos, they provide two databases for comparing banking efficiency: one that contains the embryo collection results from approximately 11,000 rat embryo donors (111 models) and another that contains the embryo collection results from 4,023 mouse embryo donors (57 induced mutant models). The genotype of donor females affected the efficiency of embryo collection in two ways. First, the proportion of females yielding embryos varied markedly among genotypes (rats: 16-100 %, mean =71 %; mice: 24-95 %, mean =65 %). Second, the mean number of embryos recovered from females yielding embryos varied considerably (rats: 4-10.6, mean =7.8; mice 5.3-32.2, mean =13.7). Genotype also affected the efficiency of rederivation of banked rat and mouse embryos models by embryo transfer. For rats, thawed embryos (n =684) from 33 genotypes were transferred into 66 recipient females (pregnancy rate, 78 %). The average rate of developing live newborns for individual rat genotypes was 30 % with a range of 10 to 58 %. For mice, thawed embryos (n =2,064) from 59 genotypes were transferred into 119 pseudopregnant females (pregnancy rate: 76 %). The average rate of development of individual mouse genotypes was 33 % with a range of 11 to 53 %. This analysis demonstrates that genotype is an important consideration when planning embryo banking programs.     

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.     

tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.     

Efficient production of intersubspecific hybrid mice and embryonic stem cells by intracytoplasmic sperm injection

Recently, mice and embryonic stem (ES) cells with allelic polymorphisms have been used extensively in the field of genetics and developmental biology. In this study, we examined whether intersubspecific hybrid mice and ES cells with these genotypes can be efficiently produced by intracytoplasmic sperm injection (ICSI). Frozen-thawed spermatozoa from wild-derived strains, JF1 (Mus musculus molossinus), MSM (M. m. molossinus), HMI (M. m. castaneus), and SWN (M. m. spp.), were directly injected into mature oocytes from laboratory mice ([C57BL/6 x DBA2]F1; M. m. domesticus). The in vitro and in vivo developmental capacity of F1 embryos was not significantly different among the groups (P > 0.05), and term offspring were efficiently obtained in all groups (27%-34% of transferred embryos). However, the mean body and placental weights of the offspring differed significantly with genotype (P < 5 x 10(-10)), with the HMI hybrid greatest in both body and placental weights. In an application study using these F1 offspring, we analyzed their mitochondrial DNA using intersubspecific polymorphisms and found the consistent disappearance of sperm mitochondrial DNA in the F1 progeny. In a second series of experiments, we generated F1 blastocysts by injecting MSM spermatozoa into C57BL/6 oocytes and used them to generate hybrid ES cell lines. The ES cell lines were established at a high efficiency (9 lines from 20 blastocysts) and their allelic polymorphisms were confirmed. Thus, ICSI using cryopreserved spermatozoa allows the efficient and immediate production of a number of F1 hybrid mice and ES cell lines, which can be used for polymorphic analysis of mouse genetics.     

A 15 nucleotide deletion mutation in coding region of the RIG-I lowers grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus

RIG-I (Retinoic acid-inducible gene I) is a pivotal receptor that detects numerous RNA and DNA viruses and plays crucial roles in the induction of type I interferons. In the present study, a deletion mutation in CiRIG-I (Ctenopharyngodon idella RIG-I) coding region was detected, its association with resistance/susceptibility to grass carp reovirus (GCRV) was examined, and possible mechanism was analyzed. A 15-bp deletion mutation was found, and the mutation results in a deletion of five amino acids. To investigate the genotypes and alleles, the relevant PCR products were electrophoresed on 2.5% agarose gel. Three genotypes and two alleles were discovered. The general allele was named as A and the deletion mutation allele was named as B. The deletion mutation cancels a predicted phosphorylation site and changes the secondary structure and the probability of peroxisomal targeting signal 1 in CiRIG-I. To explore the correlation between these genotypes and the resistance of grass carp to GCRV, a challenge experiment was carried out. The cumulative mortality in genotype AA (40.70%) and AB (52.73%) was significantly lower than that in genotype BB (71.43%) (P = 0.032). The result demonstrated that genotype AA and AB were resistant to GCRV, while genotype BB was susceptible. The 15-bp deletion mutation lowers the resistance of grass carp to GCRV. This result might provide a potential genetic marker for further investigation of selective breeding of resistant grass carp to GCRV.     

北京黑猪FSHb 亚基基因的多态性与繁殖性状的关联分析

本研究以北京黑猪为研究对象, 以FSHb 亚基基因为产仔性状的候选基因, 分别采用PCR产物直接电泳和PCR-RFLP方法来检测FSHβ亚基基因2个位点的多态性。结合测序发现: FSHb-1位点上, 北京黑猪BB型的134与135 bp (D00621序列的6 473与6 474 bp) 之间插入273 bp的片段而产生多态, 序列分析表明该插入片段为一典型的逆转座子, 在插入片段中还发现了一个RNA 聚合酶Ⅲ内部启动子; FSHb-2位点上, 由于扩增片段173 bp处存在C→T的突变, 使得HaeⅢ酶切位点消失而产生多态; 2个位点的A、B等位基因在北京黑猪群体中都有分布, 且处于低度多态。χ2适合性检验结果表明, 该群体在这2个位点的突变都达到Hardy-Weinberg平衡状态 (P>0.05)。用SAS 8.2 软件最小二乘法拟合线性模型, 将基因座不同基因型与繁殖性状总产仔数 (TNB)、产活仔数 (NBA) 和出生重 (WB) 进行了关联分析, 结果表明: 就初产母猪而言, FSHb-1位点上, AA型比AB和BB型个体的TNB分别多0.96头和1.85头 (P＜0.05), AA和AB型比BB型个体的NBA分别多0.95头和1.69头(P＜0.05)。FSHb-2位点上, AA型比AB型和BB型个体的TNB分别多1.57头和2.15头 (P＜0.05); AA和AB型比BB型个体的NBA分别多1.00头和0.94头 (P＜0.05); 就经产母猪而言, FSHb-2位点上, AA型个体的WB比BB型的WB重0.25 kg (P＜0.05)。全部群体的FSHb-1位点的A等位基因和初产母猪FSHb-2位点的A等位基因对TNB、NBA和WB表现为正效应。