The Bombyx mori genome: analysis by DNA reassociation kinetics

The size and nucleotide sequence complexity of the Bombyx mori genome has been determined from the kinetics of reassociation of its DNA. Nonrepeated DNA comprises 55% of the genome, and the remainder is divided equally between sequences repeated roughly 500 and 50000 times. Non-repeated sequence DNA virtually free of repeated sequences was prepared by partial reassociation and subsequent fractionation on hydroxyapatite. The nucleotide sequence complexity of this component was determined relative to DNA from B. subtilis and E. coli. After correction for the size of the repeated sequence fraction, the DNA content of the Bombyx mori genome was calculated to be 0.53±0.02×10^?12 g. This value compares favorably with the DNA content of haploid B. mori spermatids and mature sperm determined cytophotometrically by Rasch (1973).     

Lampbrush-type chromosomes in the primary nucleus of the green alga Acetabularia mediterranea

Structures with a lampbrush-chromosome-like morphology are described in the nucleoplasm of primary nuclei of the green alga, Acetabularia mediterranea, by light and electron microscopy in sections of cells fixed in situ and in spread preparations of isolated nuclear components. These chromosomes reveal typical loops (up to 20 μm long), chromomere-like nodules (1–2 μm in diameter), and 2–4 μm large axial globules. Associations of some of these chromosomes with nucleolar structures and with the nuclear envelope are also recognized. The light microscopically identified loops are correlated with distinct fibrillogranular structures observed in the thin sections and with the very long matrix units seen in the spreadpreparations. The similarity of these structures to the lampbrush chromosomes of various animal cell types, all exclusively stages of meiotic prophase, is discussed as well as the possible relation of the appearance of lampbrush chromosomes to a defined phase of the vegetative growth of this alga.     

Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme–substrate complexes

Tyrosyl-tRNA synthetase from Mycobacterium tuberculosis (MtTyrRS) is an enzyme that belongs to class I of aminoacyl-tRNA synthetases, which catalyze the attachment of l-tyrosine to its cognate tRNATyr in the preribosomal step of protein synthesis. MtTyrRS is incapable of cross-recognition and aminoacylation of human cytoplasmic tRNATyr, so this enzyme may be a promising target for development of novel selective inhibitors as putative antituberculosis drugs. As a class I aminoacyl-tRNA synthetase, MtTyrRS contains the HIGH-like and KFGKS catalytic motifs that catalyze amino acid activation with ATP. In this study, the conformational mobility of MtTyrRS catalytic KFGKS loop was analyzed by 100-ns all-atoms molecular dynamics simulations of the free enzyme and its complexes with different substrates: tyrosine, ATP, and the tyrosyl–adenylate intermediate. It was shown that in the closed state of the active site, the KFGKS loop, readily adopts different stable conformations depending on the type of bound substrate. Molecular dynamics simulations revealed that the closed state of the loop is stabilized by dynamic formation of two antiparallel β-sheets at flanking ends which hold the KFGKS fragment inside the active center. Prevention of β-sheet formation by introducing point mutations in the loop sequence results in a rapid (<20 ns) transition of the loop from its functional “closed” M-like structure to an inactive “open” O-like structure, i.e. rapid diffusion of the catalytic loop outside the active site. The flexibility and rapid dynamics of the wild-type aaRS catalytic loop structure are crucial for formation of protein–substrate interactions and subsequently for overall enzyme functional activity.     

Replicating DNA molecules from fertilized eggs of Cochliomyia hominivorax (Diptera)

DNA samples were prepared from the early embryos of the screwworm, Cochliomyia hominivorax. When they were examined under the electron microscope, linear DNA molecules containing one or more replicating loops arrayed tandemly were abundantly observed. The average size of replicating loops is 0.83 μ. Further, the average distance between center-to-center of adjacent loops was found to be 2.3 μ. Both these numbers are considerably smaller than those of D. melanogaster. Comparisons between the developmental time and the replication pattern of these two insects are discussed.     

Expression Analysis of Bombyx mori Bidensovirus Structural Proteins and Assembly of Virus-like Particles in Insect Cells

Bombyx mori bidensovirus (BmBDV) is a new designated species of the new genus Bidensovirus in the new family Bidnaviridae, which contains two single-stranded linear DNAs (VD1 and VD2) and causes the chronic densonucleosis disease of silkworm. Previous researches revealed that VD1-ORF3 encodes the major structural proteins VPs. In this work, through western blot, we found that VPs expressed from 48 h post-inoculation and kept increasing until 120 h post-inoculation in midgut of Bombyx mori. In order to further investigate the translation of vp gene, the ORFs (vp1 and vp2) of the VP started just up-stream of the first two candidate initiation codons were expressed in Sf9 cells by a baculovirus expression system. The expression products were purified by gradient density centrifugation and analyzed by Western blot and electron microscopy. The results showed that the expressions of vp1 yielded three proteins (VP1, VP1′, and VP2), which are the same with the viral VPs expression in midgut of Bombyx mori, and vp2 generated two VPs with the molecular weights of about 51 kDa (VP2) and 37 kDa. The observation by electron microscopy indicated that these VPs can auto-assemble into virus-like particles that could not be distinguished from virus particles. These findings will provide materials for studying the structure of BmBDV and be helpful in the studies on BmBDV-based disease in silkworms.     

Tritium labelling and cytochemistry of extra DNA in Acheta

Females of Acheta domesticus were injected with H³-thymidine and H³-uridine at various stages of development in order to study DNA and RNA synthesis in the DNA body present in the oocytes. Staining with alkaline fast green, azure B and the Feulgen reaction were employed as cytochemical tests. The following main results were obtained.

1. The DNA body appears in the oogonia at interphase as a Feulgen positive spherical structure 2 microns in diameter and is seen in subsequent mitotic divisions as a slightly smaller structure of variable shape. H³-thymidine autoradiography discloses that the DNA present in this body is synthesised at a different time from the chromosomal DNA.
2. At interphase and during the early prophase of meiosis the DNA body increases in size becoming a large Feulgen positive sphere 6 microns in diameter. Small nucleoli are present within this body. The DNA of the body is complexed with histone as revealed by alkaline fast green staining. H³-thymidine labelling discloses that it is at these stages that the bulk of the DNA synthesis takes place in the body.
3. Every oocyte contains a DNA body, and no body of comparable size or shape seems to be present in the male meiotic prophase.
4. At pachytene and diplotene the DNA body acquires the appearance of a “puff”. Two zones can be distinguished inside the DNA body: (1) an inner core of DNA and an outer shell of RNA. The inner core is Feulgen positive and stains light green with azure B, the outer shell is Feulgen negative and stains purple-violet with azure B, as does the cytoplasm. From the inner DNA core many Feulgen positive fibrils radiate into the outer RNA shell. These fibrils appear unstained or slightly greenish with Azure B, forming a transparent network in a purple-violet background. This gives the body the typical appearance of a “puff”. H³-uridine incorporation reveals that the RNA synthesis occurs in the outer RNA shell of the body and in the chromosomes. RNase treatment removes the H³-uridine incorporated into these regions.
5. At the end of diplotene the DNA body starts to disintegrate. The DNA core breaks up into minor components and the outer RNA zone also begins to disintegrate. By late diplotene the whole body has vanished, releasing DNA, histone and RNA into the nucleus. Subsequently the nuclear envelope disintegrates as it regularly does at the end of prophase of meiosis.
6. The simplest interpretation of the above results is that the DNA body represents hundreds of copies of the genes of the nucleolar organizing region.

     

Development of a cell-based assay for ecdysteroid quantification using an early ecdysteroid-inducible gene promoter

Ecdysteroids, primarily 20-hydroxyecdysone (20E) and ecdysone (E), are steroid hormones that regulate various developmental and physiological processes in insects. Commonly, immunoassays are used to quantify ecdysteroid titers of insects. However, the antibodies used in these assays react not only with 20E and E but often also with their inactive reserves and metabolites, and thus require purification before they can be quantified precisely. Here, we developed a simple cell-based method to quantify only the hormonally active ecdysteroids using newly established cells harboring the firefly luciferase gene under the control of the ecdysteroid-inducible promoter of the E75A gene of the silkworm Bombyx mori L. These cells also constitutively expressed the Renilla luciferase gene using the baculovirus ie2 promoter for internal reference. This cell-based method detected hormonally active ecdysteroids with significantly higher sensitivity than their inactive metabolites. Hemolymph ecdysteroid titers, determined using a dual luciferase assay after exposing these cells to crude extracts of B. mori larval and pupal hemolymph, agreed well with the sum of the 20E and E titers, which were quantified individually using a radioimmunoassay after they had been separated by HPLC. Thus, this method is very useful for quantifying the ecdysteroid titers of insects, particularly when the samples contain large amounts of ecdysteroid reserves and metabolites.     

Mapping by synthetic peptides of the binding sites for acetylcholine receptor on α-bungarotoxin

A set of seven peptides constituting the various loops and most of the surface areas of α-bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bindTorpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1–16, 26–41, and 45–59, were able to bind¹²⁵I-labeled AChR and, conversely,¹²⁵I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1–16, 26–41, and 45–59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45–47).