Susceptibility of inbred mouse strains to infection with three species of Metagonimus prevalent in the Republic of Korea

Susceptibility of inbred mouse strains to Metagonimus yokogawai, Metagonimus miyatai, and Metagonimus takahashii infections was studied using BALB/c, ddY, C57BL/6J, C3H/HeN, and A/J mice, with H-2 haplotypes d, s, b, k, and a, respectively. Two hundred metacercariae were orally fed to each mouse, and the worm recovery rates (WRR), worm dimensions, and intrauterine egg numbers were measured at days 3, 7, 14, 21, and 28 postinfection (PI). On day 14 PI, the WRR of M. yokogawai was highest in ddY mice (average, 62.2%); those of M. miyatai and M. takahashii were highest in ddY (19.5%) and BALB/c mice (10.4%), respectively; worm maturation was best in C3H/HeN (M. yokogawai), C57BL/6J (M. miyatai), and ddY mice (M. takahashii). All mouse strains showed higher susceptibility to infection with M. yokogawai than with M. miyatai or M. takahashii. The results show that susceptibility of mice to Metagonimus infection varies according to mouse strain and parasite species but is suggested to be independent of the mouse H-2 haplotype.     

Sequencing of simple sequence repeat anchored polymerase chain reaction amplification products of Biomphalaria glabrata

Simple sequence repeat anchored polymerase chain reaction amplification (SSR-PCR) is a genetic typing technique based on primers anchored at the 5' or 3' ends of microsatellites, at high primer annealing temperatures. This technique has already been used in studies of genetic variability of several organisms, using different primer designs. In order to conduct a detailed study of the SSR-PCR genomic targets, we cloned and sequenced 20 unique amplification products of two commonly used primers, CAA(CT)6 and (CA)8RY, using Biomphalaria glabrata genomic DNA as template. The sequences obtained were novel B. glabrata genomic sequences. It was observed that 15 clones contained microsatellites between priming sites. Out of 40 clones, seven contained complex sequence repetitions. One of the repeats that appeared in six of the amplified fragments generated a single band in Southern analysis, indicating that the sequence was not widespread in the genome. Most of the annealing sites for the CAA(CT)6 primer contained only the six repeats found within the primer sequence. In conclusion, SSR-PCR is a useful genotyping technique. However, the premise of the SSR-PCR technique, verified with the CAA(CT)6 primer, could not be supported since the amplification products did not result necessarily from microsatellite loci amplification.     

A numerical taxonomic study on heterophyid trematodes

A numerical taxonomy was studied on a group of heterophyid trematodes and analysis was made on the following species: Metagonimus yokogawai (3 OTU, Operational Taxonomic Unit), Metagonimus Miyata Type (3 OTU), Metagonimus takahashii (2 OTU), Heterophyes dispar (2 OTU), Heterophyes heterophyes (1 OTU), Heterophyes nocens (2 OTU), Heterophyopsis continua (1 OTU), Pygidiopsis summa (3 OTU), Stellantchasmus falcatus (2 OTU) and Stictodora lari (2 OTU). Twenty-six morphological characters were measured and their values were expressed as relative ratios. Similarity and correlation matrix among each individuals were calculated. Clustering analysis by Ward's method and factor analysis were performed using the SAS (Statistical Analysis System) package. As a result, the groups belonging to the genus of Metagonimus were divided into three phenons (Metagonimus yokogawai, Metagonimus Miyata Type, M. takahashii), and Metagonimus Miyata Type was classified as the level of subspecies of M. takahashii. The groups belonging to the genus Heterophyes were clearly divided into three phenons (Heterophyes dispar, H. heterophyes, H. nocens), and H. nocens was classified as not a subspecies level of H. heterophyes but a distinct species. Other species were classified as distinct phenons. From these results, the application of numerical taxonomy on trematode classification is considered to be a great aid to determine the limit of taxa.     

Genetic Analyses of Casuarinas Using ISSR and FISSR Markers

Inter simple sequence repeat polymerase chain reaction (ISSR-PCR) was used for the genetic analysis of the six species of Allocasuarina, five species of Casuarina and 12 superior performing selections of C. equisetifolia L. We also fingerprinted C. equisetifolia L. selections using Fluorescent-ISSR-PCR (FISSR-PCR), an improvised ISSR-PCR assay. The ISSR analysis provided information on the frequency of various simple sequence repeats in the casuarina genome. The di-nucleotide repeats were more common, among which (CA)n and its complementary nucleotide (GT),, repeat motifs amplified relatively higher number of bands with an average of 6.0+/-3.5 and 6.3+/-1.8 respectively. Eleven species of casuarinas were amplified with 10 primers anchored either at 5' or 3' end. A total of 253 PCR products were obtained and all were polymorphic, out of which 48 were specific to Allocasuarina and 36 were specific to Casuarina genus. Genetic similarity among the species was 0.251. A UPGMA dendrogram grouped all the Casuarina species together. The 12 superior performing selections of C. equisetifolia L. produced 57 polymorphic ISSR markers while the FISSR assay revealed 105 polymorphic markers. The primer CRR(ATT)4 distinguished all the selections. DNA profiles obtained with ISSR and FISSR assays would serve as a reference library for the establishment of clonal identity in casuarinas.     

Reverse random amplified microsatellite polymorphism reveals enhanced polymorphisms in the 3' end of simple sequence repeats in the pepper genome

Microsatellites or simple sequence repeats (SSR) are widely distributed in eukaryotic genomes and are informative genetic markers. Despite many advantages of SSR markers such as a high degree of allelic polymorphisms, co-dominant inheritance, multi-allelism, and genome-wide coverage in various plant species, they also have shortcomings such as low polymorphic rates between genetically close lines, especially in Capsicum annuum. We developed an alternative technique to SSR by normalizing and alternating anchored primers in random amplified microsatellite polymorphisms (RAMP). This technique, designated reverse random amplified microsatellite polymorphism (rRAMP), allows the detection of nucleotide variation in the 3' region flanking an SSR using normalized anchored and random primer combinations. The reproducibility and frequency of polymorphic loci in rRAMP was vigorously enhanced by translocation of the 5' anchor of repeat sequences to the 3' end position and selective use of moderate arbitrary primers. In our study, the PCR banding pattern of rRAMP was highly dependent on the frequency of repeat motifs and primer combinations with random primers. Linkage analysis showed that rRAMP markers were well scattered on an intra-specific pepper map. Based on these results, we suggest that this technique is useful for studying genetic diversity, molecular fingerprinting, and rapidly constructing molecular maps for diverse plant species.     

Distorted Segregations of RFLP Markers and Their Distribution on Chromosomes in an indica/japonica F2 Population of Rice (Oryza sativa L.)

he segregation ratio of RFLP markers in an F2 population from indica "Zhaiyeqing 8” and japonica "Jingxi 17' of rice (Oryza sativa L., 2n= 24) was studied using 54 RFLP markers distributed on 12 chromosomes. Distorted segregation was found in 25.9% of the marker tested, which was indicated by significant deviation from the expected Mendelian segregation ratio ( I: 2: 1) at 5% or 1% level. Among the three RFLP genotypes of the F2 population “Zhaiyeqing” 8 genotype was significantly more than the expected, and its gene frequency was up to 52.1 %. Three positions for distorted segregation were found on chromosome 3 (RG227-RG369), 7 (RG678-RG511-RG528) and 12 (RG463-RG323). These positions could be related to gametophyte loci responsible for the distortion.     

勒氏笛鲷微卫星位点的筛选及特征分析

采用PCR法快速筛选勒氏笛鲷(Lutjanus russelli)基因组文库, 以获得(CA)_n微卫星位点。勒氏笛鲷基因组DNA经限制性内切酶HaeⅢ+ DraⅠ双酶切后, 连接T-载体克隆, 构建基因组文库。以通用引物M13+/-与重复序列引物(CA)15对基因组文库进行筛选, 二次筛选后得到121个可能含有微卫星位点的阳性克隆。进行序列测定, 共获得53个CA(n≥7)重复序列, 重复次数主要分布于7~15(80.77%)。在所得微卫星序列中, 重复单元除CA外, 还观察到单碱基、三碱基、四碱基、五碱基重复单元。根据侧翼序列设计48对引物, 通过优化PCR反应条件, 可获得清晰可重复的目的条带。研究旨在为勒氏笛鲷遗传多样性研究及遗传图谱的构建等奠定基础, 为勒氏笛鲷资源的合理开发利用提供参考。     

Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica

The tyrosinase inhibitory activity of methanol extracts of the leaves of 39 plant species growing on the seashore of Iriomote island (Okinawa, Japan) was investigated. The extracts of Hibiscus tiliaceus, Carex pumila, and Garcinia subelliptica showed potent activity among them. The inhibitors in the extract of Garcinia subelliptica were purified by assay-guided fractionation to give two biflavonoids. These were known compounds (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone and 5,7,4',5',7',3',4'-heptahydroxy[3-8'] biflavanone), although their strong inhibitory activity toward tyrosinase is revealed for the first time in this work. One of these biflavonoids (2R,3S-5,7,4',5',7',3',4'-heptahydroxy flavanone[3-8'] flavone) showed much stronger activity (IC50 2.5 microM) than that of kojic acid (IC50 9.1 microM) when L-tyrosine was used as the substrate.     

Prolyl endopeptidase inhibitors from Syzygium samarangense (Blume) Merr. & L. M. Perry

Compounds isolated from the hexane extract of the leaves of Syzygium samarangense (Blume) Merr. & L. M. Perry were tested for inhibitory activity against the following serine proteases: trypsin, thrombin and prolyl endopeptidase. The compounds were identified as an intractable mixture of alpha-carotene and beta-carotene (1), lupeol (2), betulin (3), epi-betulinic acid (4), 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (5), 2'-hydroxy-4',6'-dimethoxy-3'-methylchalcone (6), 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (7), 2',4'-dihydroxy-6'-methoxy-3'-methyldihydrochalcone (8) and 7-hydroxy-5-methoxy-6,8-dimethylflavanone (9). Hydrogenation of compounds 5, 6 and 7 yielded compound 8, 2'-hydroxy-4',6'-dimethoxy-3'-methyldihydrochalcone (10) and 2',4'-dihydroxy-6'-methoxy-3',5'-dimethyldihydrochalcone (11), respectively. The hydrogenated products of compounds 6 and 7 were also tested for enzyme inhibitory activity. In addition, beta-sitosterol (12) and beta-D-sitosterylglucoside (13) were also isolated. This is the first report of the isolation of compounds 1-6, 8 and 13 from this plant. Compounds 3-8 and 10 exhibited significant and selective inhibition against prolyl endopeptidase among three serine proteases. This is the first report of this kind of activity for all these compounds.     

Isolation of trinucleotide microsatellite markers for Mystus nemurus

Twelve single locus trinucleotide microsatellite markers were developed to characterize the Asian river catfish, Mystus nemurus, an important food fish in South East Asia. They were obtained by using a rapid method namely the 5' anchored PCR enrichment protocol. The specific primers were designed to flank the repeat sequences and these were subsequently used to characterize 90 unrelated fish from Malaysia. The number of alleles per locus ranged from 2 (MnVj2-281) to 12 (MnBp8-4-43b) while the levels of heterozygosity ranged from 0.0444 (MnVj2-1-19) to 0.7458 (MnVj2-291).     

Detection of hyperthermophilic archaea of the genus Desulforococcus by hybridization with oligonucleotide probes

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining of PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a Crenarchaeota-specific primer pair designed: Cren 7F (5'-TTCCGGTTGATCCYGCCGGACC-3') and Cren 518R (5'-GCTGGTWTTACCGCGGCGGCTGA-3'). The PCR products were hybridized with Dig-11-dUTP-labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5'-CGTTAACYCCYGCCACACC-3) and its species D. mobilis (Dco_mob 198, 5'-CGTTAACCCCTGCCACACC-3') and D. amylolyticus (Dco_amy 198, 5'-CGTTAACCCCCGCCACACC-3'). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the species Desulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at the Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.     

Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails.     

丁坝对鱼类栖地的影响范围评估

利用磁珠富集法和5’锚定PCR法开发背瘤丽蚌的微卫星标记,将获得的多态性引物用于群体的遗传多态性分析,以期在比较两种开发微卫星标记方法的基础上同时获得一批有用的微卫星引物。从磁珠富集法获得的微卫星序列阳性克隆率为69.2%,重复次数超过10的占总数的70.2%,从设计的28对引物中筛选得到多态性引物11对,开发效率为39.3%。这11对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为4～13,观测杂合度、期望杂合度范围分别为0.205～0.738、0.566～0.839。而5’锚定PCR法获得的微卫星序列阳性克隆率为97.8%,重复次数超过10的占总数的24.7%,从设计的56对引物中筛选得到多态性引物19对,开发效率为30.4%。这19对引物用于养殖群体的遗传多样性分析,结果显示,等位基因数范围为3～10,观测杂合度、期望杂合度范围分别为0.208～0.894、0.431～0.896。实验结果表明,磁珠富集法所获微卫星序列质量高,开发微卫星标记效率较高;而5’锚定PCR法实验操作更简便,所获得的引物遗传多样性指数更高。两种方法开发的引物均可用于背瘤丽蚌和近缘种的野生种质资源遗传多样性研究。     

Staphylococcus aureus helicase but not Escherichia coli helicase stimulates S. aureus primase activity and maintains initiation specificity

Bacterial primases are essential for DNA replication due to their role in polymerizing the formation of short RNA primers repeatedly on the lagging-strand template and at least once on the leading-strand template. The ability of recombinant Staphylococcus aureus DnaG primase to utilize different single-stranded DNA templates was tested using oligonucleotides of the sequence 5'-CAGA (CA)5 XYZ (CA)3-3', where XYZ represented the variable trinucleotide. These experiments demonstrated that S. aureus primase synthesized RNA primers predominately on templates containing 5'-d(CTA)-3' or TTA and to a much lesser degree on GTA-containing templates, in contrast to results seen with the Escherichia coli DnaG primase recognition sequence 5'-d(CTG)-3'. Primer synthesis was initiated complementarily to the middle nucleotide of the recognition sequence, while the third nucleotide, an adenosine, was required to support primer synthesis but was not copied into the RNA primer. The replicative helicases from both S. aureus and E. coli were tested for their ability to stimulate either S. aureus or E. coli primase. Results showed that each bacterial helicase could only stimulate the cognate bacterial primase. In addition, S. aureus helicase stimulated the production of full-length primers, whereas E. coli helicase increased the synthesis of only short RNA polymers. These studies identified important differences between E. coli and S. aureus related to DNA replication and suggest that each bacterial primase and helicase may have adapted unique properties optimized for replication.     

鲤鱼微卫星分子标记的筛选

微卫星 (ｍｉｃｒｏｓａｔｅｌｌｉｔｅ)是近十几年来发展起来的一种新的分子标记 ,它是指以少数几个核苷酸 ( 1～ 6个 )为单位多次重复的简单序列 ,以双核苷酸重复最为常见 ,而其中又以 (ＣＡ/ＧＴ) ｎ 居多。由于微卫星在真核生物基因组中是随机分布的 ,而作为分子遗传标记又有着非常高的多态性和共显性 ,因此在构建遗传连锁图谱时备受青睐 (Ｂｒｏｏｋｅｔａｌ ,1994 )。目前 ,在人类和多种动物中已经构建了以微卫星为主的遗传连锁图谱。但在鲤鱼等水产动物的遗传连锁图谱中 ,微卫星分子标记还较少 (孙效文和梁利群 ,2 0 0 0 )。为了摸索…     

RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. III. Detection of intact primer RNA

RNA-linked DNA fragments of T7-infected Escherichiacoli were labeled with [(32)P]orthophosphate invivo. The RNA segments of the labeled fragments were isolated by degrading the DNA portion with the 3'--> 5' exonuclease intrinsic to bacteriophage T4 DNA polymerase and fractionated according to net charge by a DEAE-Sephadex A-25 column chromatography in the presence of 7 M urea. Tri-, tetra- and pentanucleotides were obtained which have ATP residues at their 5' ends. Most of the pentanucleotides had a single deoxynucleotide at the 3' end but a minor portion was totally an oligoribonucleotide. In the light of prior results, the former is a cooligomer of an intact tetraribonucleotide primer and a monodeoxynucleotide and the latter is an intact pentaribonucleotide primer. Tri- and tetraribonucleotides with ATP at the 5' ends had no deoxynucleotide at the 3' ends, therefore it is not clear if intact triribonucleotide primers are present. The 5'-terminal dinucleotides of the tetra- and pentanucleotides were mostly pppApC and a trace amount of pppApA was present.Images     

Synthesis and biophysical evaluation of minor-groove binding C-terminus modified pyrrole and imidazole triamide analogs of distamycin

Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.     

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.     

Cloning of two different 5' untranslated exons of bovine acidic fibroblast growth factor by the single strand ligation to single-stranded cDNA methodology.

In an attempt to characterize the 5' UTR of the aFGF mRNAs we used the new anchored PCR methodology, single strand ligation to ss-cDNAs (SLIC). In bovine brain and retina, two kinds of aFGF cDNA clones were isolated. They contained two alternative exons located 34 bp upstream to the translation initiation codon ATG. Taking into account the number of clones specific for each exon, the two mRNAs are expressed with the same ratio in both tissues. One of these bovine 5' UTR exons (136 bp) showed 81% identity to a human 5' UTR exon, the second one (323 bp) was 70% identical to the second human 5' UTR exon with a central region of 90 nucleotides showing 41% identity. The conservation of the splicing positions for these 5' UTR alternate exons in both bovine and human species, suggests that the overall structure of the aFGF gene is conserved in mammals. Furthermore, the conservation of the nucleotide sequences and of the localization of these 5' UTR exons suggests that these non-coding regions may be involved in the control of aFGF gene expression.