Genetics of insect hemolymph β-N-acetylglucosaminidase in the silkworm,Bombyx mori

The distribution of insect hemolymph -N-acetylglucosaminidase was investigated in the silkworm, Bombyx mori. Activity in 115 varieties was 6.92±3.22 units/ml, ranging from 1.4 to 17.0 units/ml. No enzyme-deficient individuals were observed. By selecting individuals showing either high or low enzyme activities, homozygotes were separated with activities varying 10-fold between isolates. No differences in activity of -mannosidase and -galactosidase were observed. Thus, it appears that high- or low-enzyme silkworms (High or Low lines) shared the same genetic background except for the gene concerning the activity of -N-acetylglucosaminidase. Studies on the heredity of the enzyme indicated that the synthesis of the enzyme protein was controlled by an autosomal allele. Examination by immunotitration and CM₅₂-cellulose column chromatography revealed that the difference in activity between High and Low lines was due to the amount of the active enzyme, but not to an endogeneous activator or inhibitor. Furthermore, there was no isozymic difference in -N-acetylglucosaminidase. Slab gel electrophoresis on polyacrylamide showed a species of enzyme (A) that stained more intensely in the High line. For the second species of enzyme (B), the converse was true. This evidence suggests that enzyme levels in hemolymph are under the control of a gene affording association of enzyme subunits to the active enzyme molecule.     

Genetics of insect hemolymph α-mannosidase in the silkworm,Bombyx mori

The genetics of hemolymph alpha-mannosidase was investigated in the silkworm, Bombyx mori. By selecting individuals showing either high or low enzyme activities, homozygotes were separated, with activities varying about five-fold. No differences in the activities of beta-galactosidase and beta-N-acetylglucosaminidase were observed. Thus, it seems that high- and low-enzyme silkworms (High and Low Lines) share the same genetic background except for the gene concerning the activity of alpha-mannosidase. The synthesis of the enzyme is controlled by an autosomal allele. Furthermore, expression of the gene varies from tissue to tissue, and there is no correlation between enzyme activity and growth rate. The difference in activity between High and Low lines is due to the amount of active enzyme, not to an endogeneous activator or inhibitor. There was no isozymic difference in alpha-mannosidase.     

Cell junctions in the cyst envelope in the silkworm testis,Bombyx mori linné

Summary The cell junctions of the cyst envelope in the testes of Bombyx mori were examined by electron microscopy utilizing a thin-sectioning technique following conventional fixation, tannic acid fixation and lanthanum tracer study, and also using a freeze-fracture technique. There are three kinds of junctions; septate junctions, gap junctions and tight junctions. Septate junctions are of the pleated type. Gap junctions are characterized by four electron-dense lines and three electronlucent lines in the reduced intercellular spaces seen by thin-sectioning. They are of the E type, having clusters of intramembraneous particles on the E-fracture face. The most striking finding is the frequent presence of tight junctions on the fracture planes, while focally fused outer leaflets of the junctional unit membranes are rarely detected on thin-sectioned preparations. Tight junctions are characterized by branching zigzag ridges on the P-fracture face and complementary grooves on the E-fracture face. It is proposed that tight junctions are new morphological evidence of blood-germ cell barrier in an insect. Acknowledgements: For helpful assistance the authors are indebted to their colleagues Miss N. Minemoto, Miss H. Kiyotake and Mr. Y. Goto     

Precise A•T to G•C base editing in the zebrafish genome

Background

Base editors are a class of genome editing tools with the ability to efficiently induce point mutations in genomic DNA, without inducing double-strand breaks or relying on homology-direct repair as in other such technologies. Recently, adenine base editors (ABEs) have been developed to mediate the conversion of A?T to G?C in genomic DNA of human cells, mice, and plants. Here, we investigated the activity and efficiency of several adenine base editors in zebrafish and showed that base editing can be used to create new models of pathogenic diseases caused by point mutations.

Results

The original ABE7.10 exhibits almost no activity in zebrafish. After codon optimization, we found that a zABE7.10 variant could induce targeted conversion of adenine to guanine in zebrafish at multiple tested genomic loci, and all the target sites showed a high rate of germline targeting efficiency. Furthermore, using this system, we established a zebrafish model of 5q-Syndrome that contained a new point mutation in rps14. The further modification of zABE7.10 by a bipartite nuclear localization signals (bpNLS) resulted in 1.96-fold average improvement in ABE-mediated editing efficiency at four sites.

Conclusions

Collectively, this system, designated as zABE7.10, provides a strategy to perform A?T to G?C base editing in zebrafish and enhances its capacity to model human diseases.

     

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.     

Functional and pharmacological characterization of a β-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding a seven-transmembrane receptor was cloned from the nervous tissues of silkworm (Bombyx mori) larvae. Sequence analysis indicated that the gene is an ortholog of CG6989, which encodes a Drosophila β-adrenergic-like octopamine (OA) receptor (DmOctβ2R). As very little information is available regarding this class of receptors, we generated a cell line that stably expressed the gene in HEK-293 cells and we then performed functional and pharmacological studies of this receptor. [³H]OA-binding assays using membrane preparations of this cell line showed that the receptor possesses a higher affinity for OA than for tyramine (TA) or dopamine (DA). The cell line elicited a bell-shaped, OA concentration-dependent increase in intracellular cAMP levels, with a maximum at 100 nM. (R)-OA was more potent than (S)-OA. TA and DA had weak or marginal effects on cAMP production. The OA receptor agonist demethylchlordimeform elicited a similar biphasic response, although the maximum response was attained at a concentration as low as 1 nM. The rank order of potency of other agonists was as follows: naphazoline > tolazoline, clonidine. Among the antagonists tested, only chlorpromazine significantly attenuated the OA-induced increase in cAMP levels. No increase in intracellular Ca²⁺ levels was observed with OA at concentrations up to 100 μM. These findings indicate that the cloned receptor is a β-adrenergic-like OA receptor with unique functional and pharmacological properties.     

Immunocytochemical localization of β-1,3-glucan recognition protein in the silkworm,Bombyx mori

Summary A monospecific antibody against -1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.     

Construction and analysis of the protein–protein interaction network for the olfactory system of the silkworm Bombyx mori

Olfaction plays an essential role in feeding and information exchange in insects. Previous studies on the olfaction of silkworms have provided a wealth of information about genes and proteins, yet, most studies have only focused on a single gene or protein related to the insect's olfaction. The aim of the current study is to determine key proteins in the olfactory system of the silkworm, and further understand protein–protein interactions (PPIs) in the olfactory system of Lepidoptera. To achieve this goal, we integrated Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses. Furthermore, we selected 585 olfactory-related proteins and constructed a (PPI) network for the olfactory system of the silkworm. Network analysis led to the identification of several key proteins, including GSTz1, LOC733095, BGIBMGA002169-TA, BGIBMGA010939-TA, GSTs2, GSTd2, Or-2, and BGIBMGA013255-TA. A comprehensive evaluation of the proteins showed that glutathione S-transferases (GSTs) had the highest ranking. GSTs also had the highest enrichment levels in GO and KEGG. In conclusion, our analysis showed that key nodes in the biological network had a significant impact on the network, and the key proteins identified via network analysis could serve as new research targets to determine their functions in olfaction.     

Identification and characterization of genes abnormally expressed in wing-deficient mutant (flügellos) of the silkworm,Bombyx mori

The wing-deficient mutant, flügellos (fl), of the silkworm lacks four wings in the pupa and the adult, due to aberrant wing morphogenesis during metamorphosis. To elucidate the mechanisms of wing-specific deficiencies in the fl mutant, we used mRNA differential display and identified five genes abnormally expressed in the fl wing discs. Northern blot and RT-PCR analyses revealed that four genes were overexpressed, but the fifth one was not transcribed in the fl wing discs. The expression level of ribosome-associated protein p40 in the fl wing discs was elevated approximately 10 times compared to the wild-type (WT) discs. Another overexpressed gene CB10 encodes a novel wing-specific protein with a putative zinc-finger motif. Overexpression of two components of extracellular matrix, cuticle protein 18 (BMCP18) and a fibrillin-like protein AD10, may result in the abnormal wing morphogenesis in the fl mutant. In contrast, a novel member of multifunctional Ca2+-binding protein annexins, designated as annexin b13 (Anx b13), was expressed dominantly in the wing discs of WT but completely repressed in the fl tissues. Strong expression of Anx b13 in wing discs during the fourth and fifth instar indicates that ANX B13 plays an important role in wing morphogenesis.     

Analysis of genetic relationship in mutant silkworm strains of Bombyx mori using inter simple sequence repeat （ISSR） markers

Amplified inter simple sequence repeats (ISSR) markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat (SSR) motifs were used in this study. A total of 113 markers were produced among 20 mutant swains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was (1.7080±0.4567), effective alleles (1.5194±0.3950) and genetic diversity (Ht) (0.2901±0.0415). The dendrogram produced using the unweighted pair group method with arithmetic means (UPGMA) and cluster analysis made using Nei's genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm swains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm swains maintained at the germplasm center.     

Molecular cloning and characterization of a recombinant Bombyx mori tyramine-β-hydroxylase in a silkworm cell line using a baculovirus expression vector system

Octopamine (OA) and tyramine (TA) are biogenic amines that act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. Tyramine-β-hydroxylase (TβH) catalyzes the biosynthesis of OA from TA. In this study, cDNA encoding Bombyx mori TβH (BmTβH) was cloned from the brain of the silkworm B. mori. The BmTβH mRNA comprised 2204 nucleotide residues and contained an open reading frame encoding 592 amino acids. The deduced amino acid sequence shared homology to several proteins belonging to the insect TβH family. Functional expression of the cloned cDNA was obtained using a B. mori baculovirus expression vector system. Western blot analysis revealed an immunoreactive band with a molecular mass of ~ 67.4 kDa. Reverse-phase high-performance liquid chromatography (HPLC) was used to identify the products formed during incubation of the enzyme reaction mixture. The optimum pH and temperature for the conversion of TA to OA were 7.5 and 25 °C, respectively. During incubation, the reaction was linear for the first 30 min at 25 °C and pH 7.5. Inhibitory experiments carried out with various concentrations of an inhibitor showed that this method can be used for screening of BmTβH inhibitors.     

Advances in the human genome project – A review

While celebrating its fifth official birthday last year it seems that the Human Genome Project (HGP) has and will continue to yield important biochemical information to mankind. It is exhilarating to think about the transition from studying genome structure to understanding genome function. The collective actions of information dessimination, technology development for efficient and faster sequencing, high-volume sequencing and developing model organisms has led to its success sofar. Various genome-wide STS-based human maps were completed in 1995, including a genetic map, a YAC map, a RH map with, and an integrated YAC-RH genetic map. These maps provide comprehensive frameworks for positioning additional loci, with the current genetic and RH maps spanning essentially 100% of the human genome and the YAC maps covering 95%. Few genes, however, have yet been localized on these framework maps. To date the Human Genome Project has experienced gratifying success. The technology and data produced by the genome project will provide a strong stimulus to broad areas of biological research and biotechnology. However, enormous challenges remain.     

Immunohistological evidence of dopamine cells in the cephalic nervous system of the silkworm Bombyx mori. Coexistence of dopamine and α endorphin-like substance in neurosecretory cells of the suboesophageal ganglion

Summary Evidence of dopamine cells in the brain and the suboesophageal ganglion of the silkworm Bombyx mori was obtained immunohistologically in larvae and pupae. From six to eight and eight (two symmetrical groups of four) immunoreactive cells are present respectively in median and lateral protocerebral areas of the brain. In the suboesophageal ganglion, two cell clusters with dopamine immunoreactivity were observed. There was no clear difference in the nature of the immunohistochemical reaction and the number of cells between diapause- and non-diapause-egg producers, in both brains and suboesophageal ganglia. By examination of adjacent sections, it was possible to show that dopamine-immunoreactive cells in larval suboesophageal ganglia also contain an endorphin-like substance.     

A homolog of the human Hermansky–Pudluck syndrome-5 (HPS5) gene is responsible for the oa larval translucent mutants in the silkworm, Bombyx mori

Normally, many granules containing uric acid accumulate in the larval integument of the silkworm, Bombyx mori. These uric acid granules cause the wild-type larval integument to be white or opaque, and the absence of these granules results in a translucent integument. Although about 30 B. mori loci governing larval translucency have been mapped, most have not been molecularly identified yet. Here, based on a structural analysis of a deletion of chromosome 14 that included the oa (aojyuku translucent) locus, we concluded that the BmHPS5 encoding a Bombyx homolog of the HPS5 subunit of biogenesis of lysosome-related organelles complex-2 is the candidate for the oa locus. Nucleotide sequence analyses of cDNAs and genomic DNAs in three mutant strains, each of which were homozygous for the respective allele of the oa locus (oa, oa ² , and oa ^v ), revealed that each mutant strain has a frame shift or a premature stop codon (caused by deletion or nonsense mutation, respectively) in the BmHPS5 gene. Our findings indicate that some genes that cause the translucent phenotype in Bombyx, some HPS-associated genes in humans, and some genes that cause mutant eye color phenotypes in Drosophila are homologous and participate in an evolutionarily conserved mechanism that leads to biogenesis of lysosome-related organelles.     

Mosaic formation by developmental loss of a chromosomal fragment in a “mottled striped” mosaic strain of the silkworm,Bombyx mori

A separable chromosomal fragment of about 2.5 Mb, which carries the larval body marking gene striped (p ^S), is present in a recessive background in the kind of genetic mosaic called mottled striped in the silkworm, Bombyx mori. The somatic loss of this chromosomal fragment during cell division gives rise to white patches (variegated pigmentation) in the dorsal black stripes of the fifth instar larva. Each larger white patch in the black p ^S stripe represents the clonal expansion of an epidermal cell that lost the fragment carrying p ^S during an early developmental stage. To gain information on the developmental history of the larval epidermal cells, we have analysed a variety of mosaic individuals showing extreme mottling patterns. Based on several common features observed in mosaic patterns, we constructed a schematic model for migration of epidermal cells, which implies that several polyclonal founding cells on each lateral side of a segment move and expand toward the dorsal mid-line. To determine the timing of loss of the fragment in half-stripe mosaics, which are completely lacking the mottled black stripe on one half of the larval body, we examined several tissues from either body side for the chromosomal fragment. Pulsed field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis showed that testes and silk glands from each side of the half-stripe mosaics (two and five individuals, respectively) contained the chromosomal fragment carrying the p ^S allele, independent of the epidermal phenotypes of the respective body half. This result suggests that loss of the chromosomal fragment leading to external half-stripe mosaics might occur, not at an early stage of development such as the first nuclear division, but rather after the progenies of epidermis and internal tissues examined here diverged from each other developmentally.