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Pathogen research abs 1 || Pathogen research abs 2 || Pathogen research abs 3 || Pathogen research abs 4 || Pathogen research abs 5 || Hormone and endocrine research abs 1 || Hormone and endocrine research abs 2 || Hormone and endocrine research abs 3 || Hormone and endocrine research abs 4 || Hormone and endocrine research abs 5 || Follicle and follicular cells research abs 1 || Interferon research abs 1 || Hemoglobin research abs || Stem cell research abs || Nucleic acid research abs

Nucleic Acids Res. 2000 Nov 1;28(21):E92.
Cre-mediated germline mosaicism: a method allowing rapid generation of several alleles of a target gene.

Holzenberger M, Lenzner C, Leneuve P, Zaoui R, Hamard G, Vaulont S, Bouc YL.

INSERM U515, Hopital Saint-Antoine, 184 rue du Fbg St-Antoine, F-75571 Paris Cedex 12, France. France. holzenberget-antoine.inserm.fr

Conditional gene targeting uses the insertion of expression cassettes for the selection of targeted embryonic stem cells. The presence of these cassettes in the final targeted chromosomal locus may affect the normal expression of the targeted gene and produce interesting knock down phenotypes. We show here that the selection cassette may then be selectively removed in vivo, using three appropriately positioned loxP sites in the targeted gene and the transgenic mouse EIIaCre. This strategy was applied to two different target genes and we demonstrated that it is reliable and reproducible. First, we generated double transgenic EIIaCre/loxP mice (F1) that showed variable degrees of mosaicism for partially CRE-recombined floxed alleles. Efficiency of EIIaCre at creating mosaicism was dependent on the target gene and on parental transmission of the transgene. The segregation of partially recombined alleles and EIIaCre transgene was obtained in the next generation using mosaic F1 males. Mosaic females were unsuitable for this purpose because they systematically generated complete excisions during oogenesis. Our strategy is applicable to other approaches based on three loxP sites. As this procedure allows generation of knock down (presence of neo), knockout (total exision of the loxP-flanked sequences) and floxed substrains (excision of the selection cassette) from a single, targeted germline mutation and in a single experiment, its use may become more widespread in conditional mutagenesis.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11058142&dopt=Abstract

Nucleic Acids Res. 2000 Nov 1;28(21):E93.
Interactions of Escherichia coli RNA with bacteriophage MS2 coat protein: genomic SELEX.

Shtatland T, Gill SC, Javornik BE, Johansson HE, Singer BS, Uhlenbeck OC, Zichi DA, Gold L.

Department of Molecular, University of Colorado, Boulder, CO 80309-0347, USA.

Genomic SELEX is a method for studying the network of nucleic acid-protein interactions within any organism. Here we report the discovery of several interesting and potentially biologically important interactions using genomic SELEX. We have found that bacteriophage MS2 coat protein binds several Escherichia coli mRNA fragments more tightly than it binds the natural, well-studied, phage mRNA site. MS2 coat protein binds mRNA fragments from rffG (involved in formation of lipopolysaccharide in the bacterial outer membrane), ebgR (lactose utilization repressor), as well as from several other genes. Genomic SELEX may yield experimentally induced artifacts, such as molecules in which the fixed sequences participate in binding. We describe several methods (annealing of oligonucleotides complementary to fixed sequences or switching fixed sequences) to eliminate some, or almost all, of these artifacts. Such methods may be useful tools for both randomized sequence SELEX and genomic SELEX.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11058143&dopt=Abstract

Nucleic Acids Res. 2000 Nov 1;28(21):E94.
PCR hot start using primers with the structure of molecular beacons (hairpin-like structure).

Kaboev OK, Luchkina LA, Tret'iakov AN, Bahrmand AR.

St Petersburg Nuclear Physics Institute, Russian Academy of Science, Gatchina 188350, Russia and Tehran Pasteur Institute, Iran. kaboemrb.pnpi.spb.ru

A new technique of PCR hot start using oligonucleotide primers with a stem-loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3'-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3'-end were added to the 5'-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem-loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11058144&dopt=Abstract

Nucleic Acids Res. 2000 Nov 1;28(21):E95.
Efficient gene targeted random mutagenesis in genetically stable Escherichia coli strains.

Fabret C, Poncet S, Danielsen S, Borchert TV, Ehrlich SD, Janniere L.

Unite de Genetique Microbienne, Domaine de Vilvert INRA, 78352 Jouy-en-Josas, France.

We describe a method to generate in vivo collections of mutants orders of magnitude larger than previously possible. The method favors accumulation of mutations in the target gene, rather than in the host chromosome. This is achieved by propagating the target gene on a plasmid, in Escherichia coli cells, within the region preferentially replicated by DNA polymerase I (Pol I), which replicates only a minor fraction of the chromosome. Mutagenesis is enhanced by a conjunction of a Pol I variant that has a low replication fidelity and the absence of the mutHLS system that corrects replication errors. The method was tested with two reporter genes, encoding lactose repressor or lipase. The proportion of mutants in the collection was estimated to reach 1% after one cycle of growth and 10% upon prolonged cell cultivation, resulting in collections of 10(12)-10(13) mutants per liter of cell culture. The extended cultivation did not affect growth properties of the cells. We suggest that our method is well suited for generating protein variants too rare to be present in the collections established by methods used previously and for isolating the genes that encode such variants by submitting the cells of the collections to appropriate selection protocols.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11058145&dopt=Abstract

Bioelectrochemistry. 2000 Sep;52(1):111-4.
On-demand electrochemical release of DNA from gold surfaces.

Wang J, Jiang M, Mukherjee B.

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces 88003, USA. joewanmsu.edu

Electrochemical quartz crystal microbalance (EQCM) is used to probe the electrochemically triggered release of nucleic acids from gold surfaces to solutions of physiological pH. The immobilization of nonthiolated DNA onto the gold surface is followed by an electrostatic desorption at -1.0 V (vs. Ag/AgCl). Steady-state frequency signals, corresponding to the removal of 261- and 644-ng/cm2 single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), respectively, were attained within 75 and 330 s. As expected for electrostatic repulsion, the amount released can be manipulated by tuning the potential. The small nonsteady-state frequency signals observed at lower potentials indicate promise for a sustained DNA release. Applicability to gold ultramicroelectrodes (12.5-microm radius) is demonstrated in connection with voltammetric blocking experiments. We expect that such on-demand electrochemical release would be a useful addition to the arsenal of nonviral gene delivery routes.

online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11059584&dopt=Abstract

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