Construction and characterization of chromosome 1B specific DNA library of wheat

Chromosome 1B was microdissected and collected from chromosome spreads of wheat (Triticum aestivum L. ‘Jing 411’), using a glass needle. DNA of the isolated chromosome was amplified in vitro by Sau3A linker adaptor-mediated polymerase chain reaction (LA-PCR). The second-round PCR products were verified by Southern hybridization using DIG-labeled genomic DNA of wheat. The results initially showed the DNA was from wheat genome. A pair of SSR primers specific to chromosome 1B was used to verify the origin of the PCR products from the isolated chromosome. The results confirmed that the PCR products originated from chromosome 1B. The second round of PCR products from chromosome 1B were cloned into plasmid pUCm-T vectors to create a chromosome-specific library, which included approximately 248 000 recombinant clones. Characterization of 100 randomly selected clones of the library showed that the insert size ranged between 0.5 and 2.0 kb, with an average of 1 kb. Randomly selected 288 clones were characterized with dot blot hybridization, of which 57.2% were medium/high copy clones and 42.8% low/single copy clones. The application of this technique to establish high-density molecular maps for chromosome 1B is discussed. Key words: Wheat, chromosome microdissection, chromosome-specific library

Characterization of the Aspergillus nidulans Septin (asp) Gene Family

Members of the septin gene family are involved in cytokinesis and the organization of new growth in organisms as diverse as yeast, fruit fly, worm, mouse, and human. Five septin genes have been cloned and sequenced from the model filamentous fungus A. nidulans. As expected, the A. nidulans septins contain the highly conserved GTP binding and coiled-coil domains seen in other septins. On the basis of hybridization of clones to a chromosome-specific library and correlation with an A. nidulans physical map, the septins are not clustered but are scattered throughout the genome. In phylogenetic analysis most fungal septins could be grouped with one of the prototypical S. cerevisiae septins, Cdc3, Cdc10, Cdc11, and Cdc12. Intron-exon structure was conserved within septin classes. The results of this study suggest that most fungal septins belong to one of four orthologous classes.

Stable variant-specific transcripts of the variant cell surface glycoprotein gene 1.8 expression site in Trypanosoma brucei.

The structure and transcriptional regulation of the 1.8 variant cell surface glycoprotein (VSG) gene expression site located on a 430-kilobase (kb) chromosome was examined in a 430-kb-chromosome-specific library. Using 32P-labeled nascent transcripts generated by nuclear run-on, we selected recombinant clones derived from the 430-kb chromosome which were coordinately activated with the 1.8 VSG gene. The results show that a repetitive region with a minimum size of 27 kb is coordinately activated with the 1.8 VSG gene. As with the 1.8 VSG gene, transcription is by RNA polymerases that are insensitive to the drug alpha-amanitin at concentrations up to 1 mg/ml. Transcription results in the generation of several stable variant-specific mRNAs. These mRNAs most likely belong to a family of repetitive expression-site-associated genes.

Stable variant-specific transcripts of the variant cell surface glycoprotein gene 1.8 expression site in Trypanosoma brucei

The structure and transcriptional regulation of the 1.8 variant cell surface glycoprotein (VSG) gene expression site located on a 430-kilobase (kb) chromosome was examined in a 430-kb-chromosome-specific library. Using 32P-labeled nascent transcripts generated by nuclear run-on, we selected recombinant clones derived from the 430-kb chromosome which were coordinately activated with the 1.8 VSG gene. The results show that a repetitive region with a minimum size of 27 kb is coordinately activated with the 1.8 VSG gene. As with the 1.8 VSG gene, transcription is by RNA polymerases that are insensitive to the drug alpha-amanitin at concentrations up to 1 mg/ml. Transcription results in the generation of several stable variant-specific mRNAs. These mRNAs most likely belong to a family of repetitive expression-site-associated genes.