Sakaki Yoshiyuki

Structural analysis of ribosomal dna homologues in nucleolus-less mutant of Xenopus laevis

Abstract Sequences homologous to the ribosomal DNA (rDNA) m a Xenopus anucleolate (nucleolus-less) mutant were analyzed by Southern blot analysis. The mutant was found to possess a variety of sequences homologous to non-transcribed spacer (NTS) and/or coding region of rDNA. 65 rDNA-homologous clones were isolated from a genomic DNA library of the mutant. All the clones showed only partial homology to the normal rDNA unit and their restriction maps differed from that of the normal rDNA unit. Based on the hybridization patterns, the rDNA-homologous clones were divided into four groups (I–IV). Structure of group IV, which most strongly hybridized to normal rDNA probe, was analyzed by nucleotide sequencing. The group IV sequence was found to contain a part of the rDNA, including Bam island, enhancer element, promoter region, external transcribed spacer, and a portion of 18 S rRNA gene. The blotting analysis suggested that the group IV sequence is specific for a particular strain of Xenopus.

Structure of the rat α2-macroglobulin-coding gene

Abstract Rat α 2 -macroglobulin ( α 2 M) is an acute-phase protein i.e., produced upon tissue inflammation. Genomic DNA clones covering the entire sequence of the α 2 M gene were isolated and characterized by restriction mapping, Southern blotting and (partial) DNA sequencing. The rat α 2 M gene is approx. 50 kb in length and consists of 36 exons ranging in size from 21 to 229 bp. Two functional domains, a bait region and a thiol ester site, are encoded by the exon 18 and 24, respectively. Several possible regulatory signals such as a TPA-inducible enhancer core, an identifier sequence, purine-pyrimidine alternative stretches and viral enhancer core sequences were identified. Several genomic DNA clones which cross-hybridized with the α 2 M cDNA probe were also identified. Sequence analysis showed that they possessed sequences identical to a part of the rat α 1 -inhibitor III cDNA and that they had a strikingly similar exon organization to the α 2 M gene.Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein, i.e., produced upon tissue inflammation. Genomic DNA clones covering the entire sequence of the alpha 2M gene were isolated and characterized by restriction mapping. Southern blotting and (partial) DNA sequencing. The rat alpha 2M gene is approx. 50 kb in length and consists of 36 exons ranging in size from 21 to 229 bp. Two functional domains, a bait region and a thiol ester site, are encoded by the exon 18 and 24, respectively. Several possible regulatory signals such as a TPA-inducible enhancer core, an identifier sequence, purine-pyrimidine alternative stretches and viral enhancer core sequences were identified. Several genomic DNA clones which cross-hybridized with the alpha 2M cDNA probe were also identified. Sequence analysis showed that they possessed sequences identical to a part of the rat alpha 1-inhibitor III cDNA and that they had a strikingly similar exon organization to the alpha 2M gene.

Efficient amplification of Drosophila simulans copia directed by high-level reverse transcriptase activity associated with copia virus-like particles

Abstract The numb er of retrotransposon copia per genome in Drosophila melanogaster cultured cells is two to three times higher than that in D. melanogaster embryo cells. Here, we have found that the genome of the related species, Drosophila simulans , contains in cultured cells more efficiently amplified copia DNA (approximately ten fold). Furthermore, we analyzed copia virus-like particles (VLPs) prepared from D. melanogaster and D. simulans cultured cells, which contain copia RNA and reverse transcriptase (RT) activity, and thus, play a major role in copia replication. The RT activity associated with the D. simulans VLPs was 25 times higher than that associated with the D. melanogaster VLPs. Taken together with the fact that copia is believed to transpose through an RNA intermediate, these results suggest that the amplification of copia DNA should relate to copia RNA-mediated transposition, and the higher RT activity associated with the D. simulans VLPs would lead to the efficient amplification of copia DNA. In a comparison between D. melanogaster and D. simulans copia nucleotide (nt) sequences, five nt substitutions, which cause the respective amino acid changes, were found in the copia RT-coding region. Polymerase chain reaction direct sequencing showed that these five substitutions are the vast majority in each Drosophila species. The substitutions, therefore, may be responsible for the high level of the RT activity associated with the D. simulans VLPs.

Two-dimensional gel electrophoretic analysis of the HindIII 1.8-kb repetitive-sequence family in the human genome

The structure and organization of a human repetitive DNA family containing the HindIII 1.8-kb repetitive sequence were studied, using two-dimensional (2D) gel electrophoresis. The HindIII 1.8-kb sequence proved to be part of a repetitive sequence about 5 kb long and interspersed on the genome. The long repetitive sequence family contained several subgroups, as based on polymorphism of the restriction site. Recombinant phages containing the long repetitive sequence were isolated from the human genomic DNA library. Heteroduplex and restriction analysis showed that the structure of the repetitive sequence carried by the phages was close to that expected from 2D gel electrophoretic analysis. The 2D gel electrophoretic analysis was shown to be a reliable and useful approach for surveying and mass analysis of repetitive sequence families.