A simple method for isolation of microsatellites from the Japanese squirrel,Sciurus lis,without constructing a genomic library

We developed a simple and easy method to isolate microsatellites without screening genomic libraries by hybridization. The method requires only three basic techniques: polymerase chain reaction, DNA cloning and sequencing. We applied this method to develop microsatellite markers for the Japanese squirrel and isolated 45 clones that contained repetitive sequences. Among the 22 clones that we tested further, we found 11 diagnostic microsatellite loci that are applicable to the molecular ecological study of Japanese squirrels.     

Microhabitat selection in the early juvenile mudskipper Boleophthalmus pectinirostris (L.)

Using laboratory choice experiments, behavioural preferences of the early juvenile mudskipper Boleophthalmus pectinirostris for temperature, salinity and sediment were observed. The temperature preference experiments were conducted in an annular chamber with a thermal gradient of 27 to 34° C, and the fish selected a mean ± s . d . temperature of 31·2 ± 0·5° C. The salinity preference experiments were conducted in an aquarium with decreasing salinity from 20 to 15, 10, 5 and 0·5, and significantly more ( P < 0·05) fish were found in water with a salinity of 5. The sediment preference experiments were tested in circular tanks which contained four types of sediment (medium sand, fine sand, muddy sand and sandy mud), and the fish showed a clear preference for sandy mud. These results indicated that early juvenile B. pectinirostris showed behavioural preference for microhabitats. Temperature and salinity probably set large-scale boundaries on distribution, but sediment should be a critical factor for determining the distribution of the mudskipper.     

Complete mitochondrial genome of the mudskipper Boleophthalmus pectinirostris (Perciformes, Gobiidae): repetitive sequences in the control region

The mudskipper, Boleophthalmus pectinirostris (Perciformes, Gobiidae), is an amphibious gobioid fish. In this paper, the complete mitochondrial genome of B. pectinirostris was firstly determined. The mitogenome (17,111 bp) comprises 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and 1 putative control region. 130-bp tandem repeat was identified in the control region, which was almost identical among the 10 individuals examined, and three different frequencies of the repeat unit (five, six or seven) were found among these individuals.     

A simple method for constructing a tagged protein

We have developed a simple method for preparing a tagged protein by PCR. With this method any protein sequence can be easily tagged. The techniques include three steps of DNA restriction, ligation and PCR. We could obtain a DNA construct containing SUMO-1 gene with His6 tag sequence with high efficiency by the next day.     

A simple method for the isolation of genomic DNA from mouse tail free of real-time PCR inhibitors

Although real-time PCR is a rapid, quantitative method for the analysis of gene and RNA levels, the presence of inhibitors in samples is an obstacle to its successful use. We have found that genomic DNA isolated from mouse tail tips using a standard proteinase K digestion method caused marked inhibition of real-time PCR. Inhibition was specific for mouse tail DNA since genomic DNA isolated from other tissue sources using the same methodology was readily amplified. We have therefore developed a nonproteinase K DNA isolation method involving the use of Chelex 100 resin. This method produces mouse tail DNA that is free of real-time PCR inhibitors.     

A rapid, simple, and effective method of constructing a randomly mutagenized plasmid library free from ligation

The QuikChange site-directed mutagenesis methodology was applied to constructing a randomly mutagenized plasmid library simply by adding manganese to the reaction mixture. This method is superior to the normally employed Pol I-type polymerase-based error-prone PCR in that (i) it does not require a subsequent ligation reaction, and (ii) there is no accumulation of mutations at the same site. alpha-Complementation analysis and subsequent sequence analyses of the lacZ alpha genes in the mutated library revealed that the mutations occurred randomly within the target gene and involved all possible base substitutions.     

Isolation of 105 microsatellite loci from an ovine genomic library enriched for microsatellites

This paper reports on the development of a small-insert (approximately 700 bp) total-genomic library for sheep specifically designed for enrichment for microsatellite (ms) loci. Four enriched libraries were prepared by amplification of the primary library with CA15, CA11, TG15 and TG11 oligonucleotide primers. A total of 11,020 clones was recovered, screened for dinucleotide repeats and over 500 positive clones sequenced. Sequence analysis indicated low clone redundancy and yielded 105 new ovine ms loci. Seventy-two percent of the new loci were found to be polymorphic in the sires of the AgResearch International Mapping Flock (IMF). The 105 new microsatellite loci increase the number of microsatellites available for sheep by >7%.     

大弹涂鱼和中华乌塘鳢肠刷状缘膜消化酶活性的比较

采用酶学分析的方法,研究了大弹涂鱼和中华乌塘鳢肠刷状缘膜的麦芽糖酶、蔗糖酶、乳糖酶、海藻糖酶、纤维二糖酶、碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等8种消化酶的活性。结果表明:1)大弹涂鱼肠Ⅱ刷状缘膜的麦芽糖酶、蔗糖酶、乳糖酶、海藻糖酶和纤维二糖酶等5种二糖酶的比活力均显著高于肠Ⅰ和肠Ⅲ(P<0·05);中华乌塘鳢肠Ⅰ刷状缘膜除乳糖酶外,其余4种二糖酶的比活力均显著高于肠Ⅱ和肠Ⅲ(P<0·05);大弹涂鱼肠Ⅲ碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等3种消化酶的比活力均显著高于肠Ⅰ和肠Ⅱ(P<0·05);中华乌塘鳢肠Ⅱ的这3种消化酶的比活力均显著高于肠Ⅰ和肠Ⅲ(P<0·05);2)大弹涂鱼各段肠刷状缘膜的5种二糖酶的比活力均显著高于中华乌塘鳢(P<0·05),前者肠刷状缘膜碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等3种消化酶的比活力整体上也稍微高于后者。由此说明:8种消化酶的活性在大弹涂鱼和中华乌塘鳢肠刷状缘膜中的分布模式明显不同,大弹涂鱼和中华乌塘鳢对二糖的消化和吸收的主要部位分别是在肠Ⅱ和肠Ⅰ,而二者对蛋白质、脂类和无机盐等营养吸收的主要部位分别是在肠Ⅲ和肠Ⅱ;大弹涂鱼和中华乌塘鳢肠刷状缘膜的5种二糖酶的活性与两者的食性关系密切,而碱性磷酸酶、氨基肽酶和γ-谷氨酰转肽酶等3种消化酶的活性与两者的食性并无密切的相关性。     

Comparative enzyme activities of the intestinal brush border membranes of the herbivorous mudskipper Boleophthalmus pectinirostris and the carnivorous Chinese black sleeper Bostrichthys sinensis

Both the mudskipper Boleophthalmus pectinirostris and Chinese black sleeper Bostrichthys sinensis live in the intertidal zone, but their feeding habits are different. The adult B.   pectinirostris is herbivorous, whereas the adult B.   sinensis is carnivorous; however, differences between the two species are not clear with regard to distribution patterns and activities of the intestinal enzymes. This study thus investigated the distribution patterns and specific activities of four disaccharidases (maltase, sucrase, lactase and trehalase), two proteases [leucine–amniopeptidase (LAP) and γ–glutamyltranspeptidase (γ-GT)] and an alkaline phosphatase (ALP) in brush border membrane fractions obtained from three purified intestinal sections of the two species. The highest activities of the four disaccharidases were found in the midgut of B.   pectinirostris and in the foregut of B. sinensis . Highest activities of the two proteases and ALP occurred in the hindgut of B.   pectinirostris and in the midgut of B. sinensis . The activities of the four disaccharidases in each intestinal section of B.   pectinirostris were significantly (P < 0.05) higher than in those of B. sinensis . The levels of LAP, γ-GT and ALP activities in the three intestinal sections varied in B.   pectinirostris and B. sinensis , suggesting that the primary regions for disaccharide digestion were in the midgut in B.   pectinirostris and in the foregut in B. sinensis , and that the hindgut of B.   pectinirostris and the midgut of B. sinensis should play important roles in final protein digestion and nutrient absorption, respectively. The activities of the four disaccharidases in the two species were well correlated with their feeding habits. However, no clear-cut correlation between the activities of the two proteases and diet could be concluded from the present study.     

A simple method for isolation of megabase DNA from cotton

A simple method for preparation of high-molecular-weight DNA from cotton was developed. This method includes two major steps, (i) isolating nuclei and (ii) embedding nuclei into agarose microbeads. DNA isolated by this procedure is larger than 5.7 Mb in size, and is suitable for physical mapping by PFGE and YAC/BAC cloning.     

苯并（a）芘对大弹涂鱼肝脏抗氧化酶活性影响的初步研究

在实验条件下,研究了不同浓度苯并(a)芘(BaP)暴露对大弹涂鱼肝脏内抗氧化酶-超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)和过氧化氢酶(CAT)活性的影响.结果表明,不同浓度的BaP暴露对抗氧化酶活性产生不同程度的影响.低浓度组(3μg     

A simple method for the isolation of actin from myxomycete plasmodia.

A new, simple method for the isolation of actin from myxomycete plasmodia has been developed. Plasmodium myosin B was incubated at 55 degrees C for 15 min in the presence of ATP or was treated with 90% acetone. By this treatment myosin was denatured completely. Actin was then extracted with a dilute ATP and cysteine solution from the heat- or acetone-treated myosin B. The method is simple and almost pure actin was obtained in high yield. The purified G-actin polymerized to F-actin on addition of 0.1 M KCl or 2 mM MgCl2. The viscosity of the purified F-actin was 8-10 dl/g. The F-actin activated muscle myosin ATPase, and actomyosin synthesized from the F-actin and muscle myosin showed superprecipitation on addition of ATP.     

Isolation, characterization and cross-species testing of microsatellites obtained from a sand smelt (Atherina boyeri) genomic library

This study reports the isolation and characterization of 11 polymorphic microsatellites from a sand smelt (Atherina boyeri) genomic library. Enrichment was performed with di-, tri- and tetranucleotide motifs following the FIASCO procedure (fast isolation by AFLP of sequences containing repeats). All loci were found to be in linkage and in Hardy-Weinberg equilibrium. This represents the first microsatellite isolation for the family Atherinidae and the isolated loci were accordingly tested on four additional species of the family: two recognized (A. presbyter and A. hepsetus) and two proposed ('punctata' and 'non-punctata' forms). Moreover their cross-species suitability on Menidia menidia, belonging to the same order but to the family Atherinopsidae, was also tested.