Shunji Natori

Cloning of gene cluster for sarcotoxin I, antibacterial proteins of Sarcophaga peregrina.

A genomic clone of sarcotoxin I was isolated. This clone contained four genes of structurally related proteins belonging to the sarcotoxin I family present in tandem array. One of these genes was sequenced and found to be the sarcotoxin IB gene. This gene contained a single intron of 95 bases.

Involvement of Sarcophaga lectin in the development of imaginal discs of Sarcophaga peregrina in an autocrine manner.

The imaginal discs of Sarcophaga were found not to develop normally in the presence of galactose, a hapten sugar of Sarcophaga lectin, or anti-Sarcophaga lectin antibody. Wing and leg discs cultured with these substances became morphologically abnormal and no imaginal discs reached the stage of terminal differentiation, even in the presence of 20-hydroxyecdysone. The development of the imaginal discs was shown to be autonomously regulated in an autocrine manner by Sarcophaga lectin; namely Sarcophaga lectin was secreted by the imaginal discs in the presence of 20-hydroxyecdysone, and the stimulus of self-induced Sarcophaga lectin seemed to be indispensable for further development of the imaginal discs. Sarcophaga lectin was originally found as a defense protein, but these results show that it plays independent roles in both defense and development.

Expression of tumor necrosis factor at a specific developmental stage of mouse embryos

We investigated the expression of the tumor necrosis factor (TNF) gene during development of mouse embryos, and observed its transient expression on Days 9 and 10 of gestation. We also detected a 25-kDa protein showing immunological cross-reactivity with mouse TNF antibody in an extract of 10-day embryos. These results suggest that TNF plays a role in mammalian ontogenesis.

Involvement of sapecin in embryonic cell proliferation of Sarcophaga peregrina (flesh fly).

Addition of antibodies against sapecin to the culture medium of NIH‐Sape‐4 cells derived from a Sarcophaga embryo greatly inhibited cell proliferation, whereas addition of sapecin stimulated cell proliferation. These results suggest that sapecin is involved in the proliferation of embryonic cells of Sarcophaga. Sapecin is known to have potent antibacterial activity, so it seems to have two different biological functions: i.e. protection against bacterial infection and stimulation of embryonic cell proliferation.

Analysis of a gene cluster for sarcotoxin II, a group of antibacterial proteins of Sarcophaga peregrina.

Sarcotoxin II is a group of antibacterial proteins of Sarcophaga peregrina (flesh fly) with related primary structures. We have cloned three genes in this family. These genes formed a tandem array with about 2-kb intervals, and one of them was present in the opposite strand. The putative amino acid sequences of the proteins encoded by these genes were very similar except for a deletion in one of them. All of the genes were found to be activated transiently in the same way when the larval body wall was injured, suggesting that the encoded proteins are acute-phase-responsive proteins for protecting the insect from bacterial infection.

Humoral factor activating the Sarcophaga lectin gene in cultured fat body

Abstract On culture of the fat body of Sarcophaga peregrina (flesh fly) larvae in modified Graces medium, the Sarcophaga lectin gene was activated only when the medium was supplemented with larval hemolymph. The expressions of the genes for the storage protein and the sarcocystatin A were not affected by supplementing the medium with the hemolymph. Thus the hemolymph contained a factor that specifically activated the Sarcophaga lectin gene. This factor seemed to be a heat-stable, low molecular weight compound that was probably not a peptide, and to activate genes in the fat body for defense proteins that are known to be expressed in response to injury of Sarcophaga larvae.

Cloning and in vitro transcription of the Sarcophaga lectin gene.

A genomic clone for the Sarcophaga lectin gene was isolated. This gene was a compact single copy gene. Two transcription initiation sites were located by S1 nuclease mapping and primer extension. However, transcription from one of these initiation sites was much greater than that from the other site under all conditions in which this gene was expressed. This gene was found to be transcribed efficiently in a nuclear extract of NIH-Sape-4 cells, an embryonic cell line of Sarcophaga synthesizing Sarcophaga lectin constitutively, but not in that of Ehrlich ascites tumor cells. These results suggested that the former extract contains a specific transcription factor(s) for this gene that is not present in the nuclear extract of Ehrlich cells.

Purification of a stage-specific and sequence-specific DNA-binding protein for the arylphorin gene of Sarcophaga peregrina

A protein that binds specifically to the nucleotide sequence ACCACAACA located at residues -247 to -255 upstream of the +1 site of the arylphorin gene of Sarcophaga peregrina was purified to homogeneity from fat body nuclei of third instar larvae. This DNA-binding protein consisted of two subunits with molecular masses of 40 kDa and 42 kDa, respectively. Accurate transcription initiation of a truncated arylphorin gene in a nuclear extract of NIH-Sape-4 cells, an embryonic cell line of Sarcophaga, was significantly enhanced in the presence of the purified DNA-binding protein.

Stage-specific detection of a DNA-binding protein for the storage protein gene of Sarcophaga peregrina

A nuclear extract of fat body prepared from third instar larvae of Sarcophaga peregrina (flesh fly) was found to contain a DNA-binding protein that specifically bound to 5-upstream region of the storage protein (arylphorin) gene. This protein was found only in larvae harvested 46 h after larval emergence, indicating that its appearance was strictly regulated by the developmental stage of the insect. Since the storage protein gene is actively transcribed in the fat body at this stage, this protein is probably a specific transcription factor for the storage protein gene. DNase I footprinting analysis showed that the nucleotide sequence of the binding site of this protein is ACCACAACA, which is located at residues -247 to -255 upstream of the +1 site. Results indicated that formation of the DNA-protein complex required Zn2+.

Preferential deadenylation of Sarcophaga lectin mRNA during its acute phase expression

The sizes of mRNAs of various defense proteins of Sarcophaga peregrina (flesh fly) were shown to become progressively shorter. We studied the shortening of the Sarcophaga lectin mRNA, because this mRNA shows acute phase expression on injury of the larvae and programmed expression in the pupal stage. Results showed that this mRNA underwent deadenylation when expressed in response to body injury, but no appreciable change in size during programmed expression in the pupal stage. During acute phase expression, the size of the poly(A) tail of the Sarcophaga lectin mRNA decreased from 130 bases to about 30 bases in 6 h. The half-lives of the forms with different sizes of poly(A) tail were almost identical.