Does Tn10 transpose via the cointegrate molecule?

Summary It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it. Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods.In the first method, 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E. coli chromosome by reciprocal recombination. The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E. coli chromosomal DNA. The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec^- strain. Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule. Then, we examined the configuration of products made by transposition of Tn10 from 55 to the E. coli chromosome. The cointegrate molecule was found in products of Tn10 transposition in a Rec⁺ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec^- strain. Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA^- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.In the second method, mobilization of pACYC177 by R388 and by R388:: Tn10 was examined. The pACYC177 plasmid was mobilized by R388::Tn10 at a frequency of 10^-4 per donor but not by R388. It occurred, in most cases, by inverse transposition of R388::Tn10 to pACYC177 forming plasmids such as pACYC177::IS10-R388-IS10. Mobilization of pACYC177 by a Tn10-mediated cointegrate in the form of pACYC177::Tn10-R388-Tn10 was not observed in crosses using a Rec^- donor. These observations also suggested that transposition of Tn10 in Rec^- cells does not occur via the cointegrate molecule.     

Life-cycle variation inPanonychus akitanus Ehara (Acarina:Tetranychidae)

The life history ofPanonychus akitanus Ehara was studied in two Hokkaido populations on dwarf bamboos. The Sapporo population overwintered both as egg and female adult onSasa senanensis, and the Tomakomai population overwintered as egg onSasa apoiensis. Mites of the Sapporo population produced four or five generations from late April to late November or early December. The eggs that had overwintered began to hatch in mid-May, and this hatching period overlapped with that of eggs laid in late April by females that had overwintered. Therefore, mites with different overwintering stages would interbreed. Most eggs that had overwintered in the Tomakomai population hatched in mid-May, and about four generations were produced before early December, when only eggs were found. The density of mites per leaf of the Sapporo population reached a maximum in early May on old leaves and in late June on new leaves, and thereafter gradually decreased. The Tomakomai population initially decreased in density after hatching in the spring, but rapidly dispersed to new leaves, reached a peak in early September, and then gradually decreased. The maximal density was about 10 times higher and the distribution was less clumped (lower values of the aggregation index,m/m) than that of the Sapporo population. This was probably because the Sapporo mites could utilize only the underside of sporadically distributed leaves which were curled by spiders, whereas the Tomakomai mites inhabited any part of the leaf undersurface of the hairy host plant. Predators observed were phytoseiid mites and larvae of gall midges. The predatory effect on the Sapporo population was not clear. In the Tomakomai population, the number of gall midges correlated with the number of spider mites better than that of phytoseiid mites.     

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases (XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10⁶.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils—analysis by chemiluminescence counting and its microscopic imaging

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca²⁺ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.     

Identification of lysine 15 at the active site in Escherichia coli glycogen synthase. Conservation of Lys-X-Gly-Gly sequence in the bacterial and mammalian enzymes

Glycogen synthases from Escherichia coli and mammalian muscle differ in many respects including regulation, sugar nucleotide specificity, and primary sequence. To compare the structure of the active sites in these enzymes, the affinity-labeling study of the E. coli enzyme was carried out using adenosine diphosphopyridoxal as the reagent. The E. coli enzyme was inactivated in a time- and dose-dependent manner when incubated with the reagent followed by sodium borohydride reduction. The inactivation was markedly protected by ADP-glucose and ADP, suggesting that the reagent was bound to the substrate-binding site. The stoichiometry of the bound reagent to the enzyme was approximately 1:1. Sequence analysis of the labeled peptide isolated from a proteolytic digest of the modified protein revealed that Lys15 is labeled. Based on the geometry of the reagent, the epsilon-amino group of this residue might be located close to the pyrophosphate moiety of ADP-glucose bound to the E. coli enzyme, like that of Lys38 in the rabbit muscle enzyme, which is labeled by uridine diphosphopyridoxal (Tagaya, M., Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 260, 6670-6676; Mahrenholz, A. M., Wang, Y., and Roach, P. J. (1988) J. Biol. Chem. 263, 10561-10567). The importance of the conserved sequence of Lys-X-Gly-Gly is discussed in connection with the glycine-rich region found in many nucleotide-binding proteins.     

Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase

Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding.     

A restriction fragment length polymorphism (RFLP) locus of rat (Rattus norvegicus) linked to theCs-1 locus of rat linkage group XIII

A novel restriction fragment length polymorphism (RFLP) in inbred rats was revealed by Southern blot analysis with a clone arbitrarily chosen from a rat genomic library as a probe. A clone named alpha 403 showed interstrain variations in the length of the EcoRI and HindIII fragments. The EcoRI fragments were either 0.7 or 3 kb, those of HindIII were either 4.5 or 5 kb, and three types were identified as combinations of those fragments in 20 inbred rat strains. These types segregated in backcross progeny as codominant alleles. The locus for the RFLP was thus named A403. Analysis of linkage between the RFLP locus and 13 other loci reveal that the A403 locus was closely linked to the Cs-1 locus (15 +/- 5.2%), which belongs to rat linkage group XIII.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139