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To characterize the structural features common to Pao-like retrotransposons, we analyzed two lambda phage clones which contain the Pao-like elements from the silkworm species Bombyx mori and B. mandarinia, and copies of Pao itself and ninja of Drosophila simulans, amplified by PCR. We previously identified two randomly amplified polymorphic DNAs (RAPDs), W-Kamikaze and W-Yamato, from B. mori and B. mandarina, which are part of two novel Pao-like retrotransposons, Kamikaze and Yamato, respectively. Complete characterization of these and other elements of this group reported here shows that Pao-like elements have common features that distinguish them from the other groups of LTR-retrotransposons. While the elements of the Ty1-copia group encode only one cysteine and histidine (Cys) motif in their gag-like region, the Pao-like elements specify three Cys motifs. The highly conserved D(35)E motif in the integrase domain of the retrotransposon polyprotein seems to be conserved in Pao-like elements, but the number of amino acid residues between D and E varies and is greater than 35. A comparison of the deduced amino acid sequences of the reverse transcriptase domain revealed the Pao-like elements are members of neither the Ty1-copia nor the gypsy-Ty3 groups. Therefore, we confirmed that the long-terminal-repeat (LTR) retrotransposons should be divided into three major groups (or families), namely the Ty1-copia, gypsy-Ty3, and Pao-like groups.

Genomic DNAs were compared between males and females of the domesticated silkworm, Bombyx mori, strains C108, C137, J137, p50, and WILD-W (constructed by crossing a wild silkworm, B. mandarina, female with a male of strain C108) by polymerase chain reaction (PCR) with 700 arbitrary 10-mer primers. Four female-specific RAPDs (W-Kabuki, W-Samurai, W-Kamikaze, and W-Yamato) were found. The sex chromosome formulas of B. mori and B. mandarina are ZW (XY) for the female and ZZ (XX) for the male. The four female-specific RAPDs are assumed to be derived from the W chromosome because the other chromosomes are shared by both sexes. A computer search for deduced amino acid sequences of these four RAPDs revealed that all of them showed homology to previously reported amino acid sequences encoded in known retrotransposable elements from various organisms.

In the silkworm, Bombyx mori, nonsusceptibility to B. mori densonucleosis virus type-1 (BmDNV-1) is controlled by a recessive gene, nsd-1 (nonsusceptibility to DNV-1), located on the twenty-first chromosome. We investigated genetic linkage between five random amplified polymorphic DNA (RAPD) markers and the +nsd-1 gene. Initially, we constructed the CSD-1 strain (nsd-1/+) which is congenic to strain C137 (nsd-1/nsd-1) for the twenty-first chromosome, starting with a female of C137 and a male of strain J137 (+nsd-1/+nsd-1). For the crossing over experiment, a female of C137 was crossed with a male (nsd-1/+) of CSD-1. Segregation analysis showed that the most closely linked RAPD marker mapped 3.0 cM distant from +nsd-1. A more specific marker for +nsd-1 was made by converting this RAPD band into a sequence characterized amplified region (SCAR) using a series of newly designed primer pairs based on its DNA sequence.