Phenoloxidase (PO) has been shown to be present in both invertebrates and verstebrates, yet little is known about the PO in Branchiostoma belcherisi tsingtauense. The present study demonstrated that the PO activity existedhistochemically the epithelil cells of the gill bar and intetne of adult animal, producing brown to black melanin deposits. Ultrastructural examination revealed that PO reaction products were localixed in the cytoplasmic granules of the epidermal cells, the epithlial cells of gill bar and intestine and most of them were electron-dense and homogenous. Frequently, the PO reaction products were also observed in the secondarylysosomes. The mucus form the body surface had little antibacterial activity against Escherichia coli, but it enhancedthe antibacterial activity of L-dopa and the enhancement was suppresed by PTUm a PO specific inhibiotor. This indicates that the active material inhibiting the E. coli growth in the mucus is PO.

The development of phenoloxidase during amphioxus embryogenesis was spectrophotometrically and histochemically studied for the first time in the present study. It was found that (1) PO activity initially appeared in the general ectoderm including the neural ectoderm and the epidermal ectoderm at the early neurula stage but not in the mesoderm or the endoderm, and (2) PO activity disappeared in the neural plate cells but remained unchanged in the epidermal cells when the neural plate was morphologically quite distinct from the rest of the ectoderm. It is apparent that PO could serve as a marker enzyme for differentiation of the neural ectoderm from the epidermal ectoderm during embryonic development of amphioxus.

Chromosome banding study remains lacking in amphioxus heretofore. This paper reports the C-band pattern of the metaphase chromosomes of amphioxus Branchiostoma belcheri tsingtauense, the first description of banded karyotype in cephalochordate. The C-banding showed that around 50% of the second chromosome pair examined was heteromorphic, while the remaining ones were homomorphic. In contrast, the other 17 pairs were all homomorphic. These broadly support our previous suggestion that the second chromosome pair is a pair of sex-chromosomes. The C-banding stained positively about 54.3% of the chromosome surface. Most of the centromeric and terminal regions in many chromosomes had positive C-bands, and some interstitial C-bands were also observed in some chromosomes. It seems clear that chromosomes of amphioxus are highly heterochromatinized. In addition, silver staining revealed a single pair of nucleolar organizer regions (NORs) located in the telomeric regions of mediumsized chromosomes. It is suggested that the number and location of NORs in amphioxus represent the primitive condition for chordates.

In thiss tudy, a member of the MyoD gene family, AmphiMDF, wasis olated from the embryos of amphioxusby degenerate PCR, followed by rapid amplification of cDNA ends (RACE). Southern blot analysis confirmed that only a single myogenic bHLH gene waspres ent in the genome of amphioxus Branchiostoma belcheri tsingtauense. Sequence and phylogenetic analyses indicated that AmphiMDF falls at the base of its vertebrate homologs. The amino acid sequence of AmphiMDF was almost equally similar to those of the four clusters of the vertebrate MyoD family. This suggests that AmphiMDF is not only the sister but also the archetype of the vertebrate myogenic bHLH genes. The scenarios to explain the origin of the vertebrate MyoD gene family from the ancestral myogenic bHLH gene like AmphiMDF are also discussed.

Amphioxus HMGB gene, AmphiHMGB, encoding a high mobility group (HMG) protein was identified from the gut cDNA library of Branchiostoma belcheri tsingtauense. Primary structural examination indicates that it is a member of HMGB family. Molecular phylogenetic analysis suggests that AmphiHMGB represents the archetype of chordate HMGB genes including HMGB1, HMGB2 and HMGB3 lthough it may have lost its C-terminal domain during evolution. Northern blotting revealed that AmphiHMGB expression was restricted mainly to guts in the adult, and AmphiHMGB transcripts were in greater abundance in gastrula and neurula though they were markedly low in 1-day larva.