Variations in BARE-1 insertion patterns in barley callus cultures

The stability of aging barley calli was investigated with the barley retroelement 1 (BARE-1) retrotransposon specific inter-retrotransposon amplified polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS medium supplemented with 3 mg/L 2,4-D and maintained on the same medium for 60 days. Ten IRAP primers were used in 25 different combinations. The similarity index between 30-day-old and 45-day-old calli was 84%; however, the similarity index between mature embryos and 45-day-old calli was 75%. These culture conditions caused BARE-1 retrotransposon alterations to appear as different band profiles. This is the first report of the use of the IRAP technique in barley in an investigation of callus development.     

Comparison of the utility of barley retrotransposon families for genetic analysis by molecular marker techniques

The Sequence-Specific Amplification Polymorphism (S-SAP) method, and the related molecular marker techniques IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism), are based on retrotransposon activity, and are increasingly widely used. However, there have been no systematic analyses of the parameters of these methods or of the utility of different retrotransposon families in producing polymorphic, scorable fingerprints. We have generated S-SAP, IRAP, and REMAP data for three barley (Hordeum vulgare L.) varieties using primers based on sequences from six retrotransposon families (BARE-1, BAGY-1, BAGY-2, Sabrina, Nikita and Sukkula). The effect of the number of selective bases on the S-SAP profiles has been examined and the profiles obtained with eight MseI+3 selective primers compared for all the elements. Polymorphisms detected in the insertion pattern of all the families show that each can be used for S-SAP. The uniqueness of each transposition event and differences in the historic activity of each family suggest that the use of multiple retrotransposon families for genetic analysis will find applications in mapping, fingerprinting, and marker-assisted selection and evolutionary studies, not only in barley and other Hordeum species and related taxa, but also more generally.     

Construction of a MluI-YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions.

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10-30% of the colonies were recombinant, as visualized by red-white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE-1 copia-like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector-insert junctions.     

Functional identification of nucleotides conferring substrate specificity to retroviral integrase reactions.

The long terminal repeats (LTRs) that flank the retroviral DNA genome play a distinct role in the integration process by acting as specific substrates for the integrase (IN). The role of LTR sequences in providing substrate recognition and specificity to integration reactions was investigated for INs from human immunodeficiency virus type 1 (HIV-1), Moloney murine leukemia virus (M-MuLV), human T-cell leukemia virus type 1 (HTLV-1), and human T-cell leukemia virus type 2 (HTLV-2). Overall, these INs required specific LTR sequences for optimal catalysis of 3'-processing reactions, as opposed to strand transfer and disintegration reactions. It is of particular note that in strand transfer reactions the sites of integration were similar among the four INs. In the 3'-processing reaction, sequence specificity for each IN was traced to the three nucleotides proximal to the conserved CA. Reactions catalyzed by M-MuLV IN were additionally influenced by upstream regions. The nucleotide requirements for optimal catalysis differed for each IN. HIV-1 IN showed a broad range of substrate specificities, while HTLV-1 IN and HTLV-2 IN had more defined sequence requirements. M-MuLV IN exhibited greater activity with the heterologous LTR substrates than with its own wild-type substrate. This finding was further substantiated by the high levels of activity catalyzed by the IN on modified M-MuLV LTRs. This work suggests that unlike the other INs examined, M-MuLV IN has evolved with an IN-LTR interaction that is suboptimal.     

大麦1H特异性CAPs标记和ASA标记的创制

选取大麦1H染色体的STS标记MWG913特异性扩增小麦,把得到的片段进行克隆.用Taq酶切分类并测序,把得到的序列同大麦的序列进行比较.依据比较结果,选取对大麦特异的内切酶,用该酶来酶切大麦、小麦、黑麦、长穗偃麦草、中间偃麦草、簇毛麦的MWG913扩增产物,获得对大麦1H染色体特异的CAPs标记.同时,依据酶切位点碱基的差异设计引物对扩增的产物进行第二次扩增,得到该位点的一对染色体特异性ASA标记.     

A crystal structure of the catalytic core domain of an avian sarcoma and leukemia virus integrase suggests an alternate dimeric assembly

Integrase (IN) is an important therapeutic target in the search for anti-Human Immunodeficiency Virus (HIV) inhibitors. This enzyme is composed of three domains and is hard to crystallize in its full form. First structural results on IN were obtained on the catalytic core domain (CCD) of the avian Rous and Sarcoma Virus strain Schmidt-Ruppin A (RSV-A) and on the CCD of HIV-1 IN. A ribonuclease-H like motif was revealed as well as a dimeric interface stabilized by two pairs of α-helices (α1/α5, α5/α1). These structural features have been validated in other structures of IN CCDs. We have determined the crystal structure of the Rous-associated virus type-1 (RAV-1) IN CCD to 1.8 Å resolution. RAV-1 IN shows a standard activity for integration and its CCD differs in sequence from that of RSV-A by a single accessible residue in position 182 (substitution A182T). Surprisingly, the CCD of RAV-1 IN associates itself with an unexpected dimeric interface characterized by three pairs of α-helices (α3/α5, α1/α1, α5/α3). A182 is not involved in this novel interface, which results from a rigid body rearrangement of the protein at its α1, α3, α5 surface. A new basic groove that is suitable for single-stranded nucleic acid binding is observed at the surface of the dimer. We have subsequently determined the structure of the mutant A182T of RAV-1 IN CCD and obtained a RSV-A IN CCD-like structure with two pairs of buried α-helices at the interface. Our results suggest that the CCD of avian INs can dimerize in more than one state. Such flexibility can further explain the multifunctionality of retroviral INs, which beside integration of dsDNA are implicated in different steps of the retroviral cycle in presence of viral ssRNA.