A TaqMan-based real-time PCR assay for the specific detection and quantification of Leuconostoc mesenteroides in meat products

A new real-time PCR procedure was developed for the specific detection and quantification of Leuconostoc mesenteroides in meat products. It is a TaqMan assay based on 23S rRNA gene targeted primers and probe. Specificity was evaluated using purified DNA from 132 strains: 102 lactic acid bacteria (LAB), including 57 reference strains and 46 food isolates, belonging to genus Leuconostoc and related genera, and 30 non-LAB strains. Quantification was linear over at least 5 log units using both serial dilutions of purified DNA and calibrated cell suspensions from Leuconostoc mesenteroides ssp. dextranicum CECT 912T. This assay was able to detect at least five genomic equivalents, using purified DNA or 59 CFU per reaction when using calibrated cell suspensions. It performed successfully when tested on an artificially inoculated meat product, with a minimum threshold of 10(4) CFU g(-1) for the accurate quantification of Leuconostoc mesenteroides.     

Immobilized Biocatalysts for the Production of Nucleosides and Nucleoside Analogues by Enzymatic Transglycosylation Reactions

The recombinant enzymes uridine phosphorylase (UP) and purine nucleoside phosphorylase (PNP) were over-expressed in high-biomass bacterial fermentations and co-immobilized, without previous purification, on epoxy-activated solid supports by covalent linkages. These preparations are efficient biocatalysts of transglycosylation reactions and have been developed for producting natural and modified nucleosides of pharmaceutical interest in the field of antiviral and antitumoral agents. The new biocatalysts described in this work are suitable for both laboratory and industrial scale applications due to the maintainance of high catalytic efficiency, thermal and solvent stability, reusability and ease of operation in batch as well as in continuous reactions.     

Influence of nitrogen/carbon ratio and complementary sugars on dextransucrase production by Leuconostoc mesenteroides NRRL B512(f).

The influence of the nitrogen/carbon ratio was studied during fermentation with Leuconostoc mesenteroides NRRL B512(f) using sucrose as substrate. The enzyme concentration was measured through activity tests and radiotracer tests with [¹⁴C]phenylalanine. In batch fermentation a slowing down in the rate of enzyme synthesis was observed with a decrease in the nitrogen/carbon ratio. Addition of pulses of nitrogen when the nitrogen/carbon ratio decreased allowed a constant production rate of enzyme and lower fermentation time. The influence of complementary sugars was also addressed. Lactose inhibited enzyme production. When galactose was used the yield was the same as in the fermentation with sucrose alone, but with a different production rate. Maltose favoured the synthesis of dextransucrase.     

A Unique Mutation of Glycoprotein Gene of the Attenuated RC-HL Strain of Rabies Virus,a Seed Virus Used for Production of Animal Vaccine in Japan

Although the RC-HL strain of rabies virus is avirulent in adult mice, the amino acid at position 333 of its G protein is arginine, which is thought to be necessary for virulence in adult mice upon intracerebral inoculation of the virus. This result suggests that besides arginine at position 333, some other positions of G protein might also be involved in determining the virulence of rabies virus.     

Characterization of the Cα-Dehydrogenase Gene Involved in the Cleavage of β-Aryl Ether by Pseudomonas paucimobilis

Cα-dehydrogenase catalyzes the oxidation of arylglycerol-β-aryl ether at the Ca-position, and therefore this process produces the specific substrate for β- etherase, which cleaves β-ary ethers (Ca carbonyl type).

Here we isolated the Ca-dehydrogenase gene (ligD) and sequenced its nucleotides. This gene contains an open reading frame of 915 bp and the deduced amino acid sequence had a homology with the ribitol dehydrogenase family. ligD is about 1 kbp upstream of the β-etherase gene (ligE).     

Molecular approaches to the study of evolution and phylogeny of the Nemertina

Molecular biological tools currently available to us are revolutionizing the way in which we can address questions in evolutionary biology. The purpose of this article is to provide an overview of molecular techniques and applications available to biologists who are interested in evolutionary studies but who have little acquaintance with molecular biology. In evolutionary biology, techniques designed to determine degree of nucleic acid similarity are in common use and will be dealt with first. Another approach, namely gene expression studies, has strong implications for evolutionary biology but generally requires substantial familiarity with molecular biological tools. Expression studies provide powerful tools for discerning processes of speciation, as in the selection of genetic variants, as well as discerning lineages, e.g., expression of specific homeobox genes during segment formation. For investigations where either nucleic acid identity or gene expression are the ultimate goal, detailed information, protocols and appropriate controls are beyond the scope of this work but, where possible, recent review articles are cited.     

小麦丛矮病毒NS蛋白基因的克隆及序列分析

利用与Ｎ蛋白ｍＲＮＡ３＇末端顺序相同的２０寡聚核苷酸引物,通过点杂交、限制性内切酶分析从小麦丛矮病毒（ＷＲＳＶ）ｃＤＮＡ文库中筛选到编码Ｎ蛋白基因下游顺序的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ片段含有一编码的４０ｋＤ蛋白的开放读框。将该读框的全长ｃＤＮＡ经ＰＣＲ扩增后,克隆到ｐＧＥＸ－３Ｘ上,在大肠杆菌ＤＥ３中用ＩＰＴＧ诱导表达,经蛋白质印迹鉴定,该基因为小麦丛矮病毒ＮＳ蛋白基因。     

小拟南芥Chitinase基因的克隆与核苷酸序列分析

采用RT-PCR扩增方法,从野生资源小拟南芥（Arabidopsis pumila)的总RNA中,克隆获得了985bp的cDNA片段,经过测序和序列分析,发现该cDNA基因包含一个完整的963bp的开放阅读框（ORF）,含有17个限制性内切酶酶切位点,核苷酸序列同源性分析表明,该基因与与Arabidopsis thaliana glycosyl hydrolase family 19(chitinase)(Atlg 05850) mRNA,complete cds(登录号NM-100466.3),Arubidopsis thaliana putative class I chitinase(Atlg05850)mRNA,complete cds(登录号AY034935）,Arabidopsi8s thaliana chitinase-like protein 1(CTL1) mRNA,CTL1-ELPlallele,complete eds(登录号）AF422178）均有94%序列同源性,Chitinase可抑制病原真菌的生长,所编码的功能蛋白在提高农作物抗病性方面具有重要意义。     

河套蜜瓜ACC合成酶cDNA片段的克隆和序列分析

1-氨基环丙烷-1-羧酸（ACC）合成酶是高等植物中乙烯生物合成的关键酶。以成熟河套蜜瓜（CucumismeloL．cvHetau）果实的RNA为模板,经反转录和PCR扩增得到预期大小的DNA片段,插入到pUC19的SmaⅠ位点后转化E.coliJM109,筛选出重组子pHMAS1。序列分析表明获得了长627bp的ACC合成酶cDNA片段。与已报道的ACC合成酶基因相应序列比较有很高的同源性．     

美国白蛾核型多角体病毒p35基因的克隆及序列分析

对美国白蛾核型多角体病毒（HycuNPV,Hyphantria cunea nucleopolyhedrovirus）p35基因的序列分析表明：HycuNPV p35编码序列900?bp, 编码299氨基酸。同源性分析表明：HycuNPV p35与BomoNPV T3、AucaNPV、SpliNPV、LeseNPV、HearNPV在核苷酸水平上为99.9%、95.7%、93.6%、80.2%和87.2%,在氨基酸水平上为99.7%、90.3%、77%、64.9%和73.2%,显示了杆状病毒p35基因在进化上的保守性。BomoNPV T3中位的H122,在HycuNPV中被R取代。推测HycuNPV p35蛋白的功能及抑制细胞凋亡的能力与BomoNPV T3 p35蛋白的相似。     

Stabilization of Crystalline Acid Carboxypeptidase from Penicillium janthinellum by Nonionic Surfactants,and Inhibition of Enzyme Activity by Anionic Compounds

The crystalline acid carboxypeptidase from Penicillium janthinellum IFO-8070 was stabilized by the addition of nonionic surfactants, such as Triton X-100, Brij 35, Span 40, and Tween 20. In the presence of these stabilizers, extremely diluted enzyme (0.3 μg/ml of 50 mm sodium acetate buffer, pH 3.7) was almost completely stable after 2 days incubation at 25°C. About 35% and 20% of the enzyme activities were activated by the addition of Triton X-100 and Brij 35, respectively. Triton X-100 completely retarded inactivation at freezing (?15°C). On the other hand, anionic surfactants of SLS and LBSA, and cationic surfactant of cetyltrimethylammonium bromide strongly inactivated the enzyme. The inhibition of the fatty acid series was roughly proportional to the molecular weight of the inhibitor. Di-, and Tri-carboxylic acids also inhibited the enzyme activity.     

Postnatal Changes in Activities of Acetylcholinesterase and Cholinesterase in the Brain of a Methylazoxymethanol-induced Microencephalic Rat

Activities of AChE and ChE per g wet weight of the cerebrum were higher in the cerebrum of the MAM-induced microencephalic rats that were the offspring of the mother rats injected on the 15th day of pregnancy with MAM-acetate than in the normal rats cerebrum. They increased for 30 days after birth and were maintained constant thereafter. The activity of AChE more increased in the cerebral subcortex than in other regions. With the increase in dose of the drug administered to mother rats, the cerebral weight of their offspring decreased gradually whereas AChE activity per g wet weight of their cerebrum increased. These findings support existence of plasticity in the cerebrum.     

Isolation and complete nucleotide sequence of the measles virus IMB-1 strain in China

The complete nucleotide sequence of the measles virus strain IMB-1, which was isolated in China, was determined. As in other measles viruses, its genome is 15,894 nucleotides in length and encodes six proteins. The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%–5% at the nucleotide sequence level. This isolate has amino acid variations over the full genome, including in the hemagglutinin and fusion genes. This report is the first to describe the full-length genome of a genotype H1 strain and provide an overview of the diversity of genetic characteristics of a circulating measles virus.     

Isolation and Complete Nucleotide Sequence of the Measles Virus IMB-1 Strain in China

The complete nucleotide sequence of the measles virus strain IMB-1,which was isolated in China,was determined.As in other measles viruses,its genome is 15,894 nucleotides in length and encodes six proteins.The full-length nucleotide sequence of the IMB-1 isolate differed from vaccine strains (including wild-type Edmonston strain) by 4%-5% at the nucleotide sequence level.This isolate has amino acid variations over the full genome,including in the hemagglutinin and fusion genes.This report is the first to de...     

Organization of the Chicken Histone Genes in a Major Gene Cluster and Generation of an Almost Complete Set of the Core Histone Protein Sequences

We present a detailed picture of the disposition of the histonegenes in the chicken genome and an almost complete set of thecore histone protein sequences. Thirty-nine histone genes, sixH1, nine H2A, eight H2B, eight H3 and eight H4, were locatedwithin a histone gene cluster of 110 kb, which was covered byfive cosmid clones and two clones. Results of our sequenceanalyses, together with those reported previously, generateda set of the core histone amino acid sequences as follows: threeH2A variants, four H2B variants,two H3 variants and an H4 protein.     

Sequence and expression of the gene for N10-formyltetrahydrofolate synthetase from Clostridium cylindrosporum.

Sau3 A and Hind III restriction fragments of Clostridium cylindrosporum genomic DNA were used to isolate clones containing 80% of the N10-H4folate synthetase gene in a 5' fragment and the remaining 20% of the gene in the 3' fragment. These fragments were joined at a common SnaB I restriction site and expressed in Escherichia coli at a level equivalent to what is normally found in C. cylindrosporum. Sequence comparisons show a large degree of homology with genes from two other clostridial species, including a thermophile. Certain conserved sequences found in the three clostridial proteins and in the N10-H4folate synthetase portion of eukaryotic C1-H4folate synthases may represent consensus sequences for nucleotide and H4folate binding.