Background: Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV)-like sequences (ERTBVs) have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response.

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.

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The translated product from the gene fragment containing the second and third domains of gramicidin S synthetase 2 was purified to an essentially homogeneous state. It showed valine- and ornithine-activating activity and the second domain was proved to be the valine-activating domain. Three mutant genes from Bacillus brevis Nagano, BI-3, E-4, and E-5 strains, which encode defective valine-activating domains of gramicidin S synthetase 2, were sequenced. By comparison with the wild-type gene, single point mutations of guanine to adenine were found at the three conserved glycine codons; the 5303rd guanine in BI-3, the 5378th guanine in E-4, and the 4967th guanine in E-5, which corresponded to codon changes of the 1768th glycine to glutamic acid and the 1793rd and the 1656th glycine to aspartic acid. Loss of valine-adenylation activity by mutation at the 1656th glycine proved the direct participation of the TSGT/STGXPKG motif in the adenylation reaction, and suggests that this glycine residue with the conserved lysine residue of the motif forms the phosphate-binding loop for ATP-binding. The 1793rd glycine is a member of the YGXTE motif which was also conserved among adenylate-forming enzymes except acetyl-CoA synthetases. The 1768th glycine residue appears to maintain the conformation of the active site for aminoacyl adenylation since this residue is retained among the adenylate-forming enzymes, though flanking regions are not conserved. These results suggest that these glycine residues are essential for adenylate formation in the antibiotic peptide synthetase family and some other adenylate-forming enzymes.

To obtain normal kits by embryo treansfer (ET) during the non-breeding season, maintenance of pregnancy was carried out by administration of sustained action progesterone (P4) in queens. Embryos were recovered six days after mating from five donor queens in which ovulation was induced by administration of eCG and hCG. The number of embryos recovered ranged from 24 to 53 (mean: 37.2 +/- 6.4) per animal and most embryos were compacted morulae. The yield of embryos was 49.0-93.3% (mean: 73.8 +/- 9.6%). As for recipients, porcine pituitary gland preparation and hCG were administered to 19 queens and estrus and ovulation were induced in 18 queens (94.7%). These queens underwent intrauterine ET of five compacted morulae and 17 cats (94.4%) were impregnated. The number of implantations was 2-5 (mean: 3.7 +/- 0.3). Among these impregnated queens, 15 cats received P4 adminstration starting on day 24 of gestation and 1-5 newborns (mean: 3.4 +/- 0.3) were obtained by normal delivery or caesarean section on day 64-69 of gestation. However, two animals that were not treated with P4 underwent spontaneous abortion about the mid gestational period. Therefore, it is possible to obtain normal kits from queens in the non-breeding season by ET with maintenance of pregnancy by P4 administration.