Molecular architecture of the kinetochore-microtubule attachment site is conserved between point and regional centromeres.

Point and regional centromeres specify a unique site on each chromosome for kinetochore assembly. The point centromere in budding yeast is a unique 150-bp DNA sequence, which supports a kinetochore with only one microtubule attachment. In contrast, regional centromeres are complex in architecture, can be up to 5 Mb in length, and typically support many kinetochore-microtubule attachments. We used quantitative fluorescence microscopy to count the number of core structural kinetochore protein complexes at the regional centromeres in fission yeast and Candida albicans. We find that the number of CENP-A nucleosomes at these centromeres reflects the number of kinetochore-microtubule attachments instead of their length. The numbers of kinetochore protein complexes per microtubule attachment are nearly identical to the numbers in a budding yeast kinetochore. These findings reveal that kinetochores with multiple microtubule attachments are mainly built by repeating a conserved structural subunit that is equivalent to a single microtubule attachment site.

Mammalian germ-line transgenesis by transposition.

Transposons have been used in invertebrates for transgenesis and insertional mutagens in genetic screens. We tested a functional transposon called Sleeping Beauty in the one-cell mouse embryo. In this report, we describe experiments in which transposon vectors were injected into one-cell mouse embryos with mRNA expressing the SB10 transposase enzyme. Molecular evidence of transposition was obtained by cloning of insertion sites from multiple transgenic mice produced by SB10 mRNA/transposon coinjection. We also demonstrate germ-line transmission and expression from transposed elements. This technique has promise as a germ-line transgenesis method in other vertebrate species and for insertional mutagenesis in the mouse.