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Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF). Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used. Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml). hEGF and Ahx-substituted hEGF, [Ahx21]hEGF, secreted into the periplasm were recovered. After treatment with H2O2, [Ahx21]-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC. Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of [Ahx21]hEGF. The three biological activities of [Ahx21]hEGF were the same as those of hEGF. From the successful production of [Ahx21]hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.

Further comparison of mitochondrial control-region DNA base sequences of 16 avian species belonging to the subfamily Phasianinae revealed the following: (i) Generalized perdicine birds (quails and partridges) are of ancient lineages. Even the closest pair, the common quail (Coturnix coturnix japonica) and the Chinese bamboo partridge (Bambusicola thoracica), maintained only 85.71% identity. (ii) The 12 species of phasianine birds previously and presently studied belonged to three distinct branches. The first branch was made exclusively of members of the genus Gallus, while the second branch was made of pheasants of the genera Phasianus, Chrysolophus, and Syrmaticus. Gallopheasants of the genus Lophura were distant cousins to these pheasants. The great argus (Argusianus argus) and peafowls of the genus Pavo constituted the third branch. The position of peacock-pheasants of the genus Polyplectron in the third branch was similar to that of the genus Lophura in the second branch. Members of the fourth phasianine branch, such as tragopans and monals, were not included in the present study. (iii) The one perdicine species, Bambusicola thoracica, was more closely related to phasianine genera Gallus and Pavo than to members of other perdicine genera. The above might indicate that Bambusicola belong to one-stem perdicine lineage that later splits into two sublineages that yielded phasianine birds, one evolving to Gallus, and the other differentiating toward Pavo and its allies.

With the aim of elucidating in greater detail the genealogical origin of the present domestic fowls of the world, we have determined mtDNA sequences of the D-loop regions for a total of 21 birds, of which 12 samples belong to red junglefowl (Gallus gallus) comprising three subspecies (six Gallus gallus gallus, three Gallus gallus spadiceus, and three Gallus gallus bankiva) and nine represent diverse domestic breeds (Gallus gallus domesticus). We also sequenced four green junglefowl (Gallus varius), two Lafayette's junglefowl (Gallus lafayettei), and one grey junglefowl (Gallus sonneratii). We then constructed a phylogenetic tree for these birds by the use of nucleotide sequences, choosing the Japanese quail (Coturnix coturnix japonica) as an outgroup. We found that a continental population of G. g. gallus was the real matriarchic origin of all the domestic poultries examined in this study. It is also of particular interest that there were no discernible differences among G. gallus subspecies; G. g. bankiva was a notable exception. This was because G. g. spadiceus and a continental population of G. g. gallus formed a single cluster in the phylogenetic tree. G. g. bankiva, on the other hand, was a distinct entity, thus deserving its subspecies status. It implies that a continental population of G. g. gallus sufficed as the monophyletic ancestor of all domestic breeds. We also discussed a possible significance of the initial dispersal pattern of the present domestic fowls, using the phylogenetic tree.

A synthetic gene for porcine secretin was ligated with Pst I-cleaved pBR322 by its flanking synthetic Pst I linkers to produce a fused protein consisting of an amino-terminal portion of beta-lactamase and an entire secretin molecule. The hybrid plasmid was transferred into competent Escherichia coli cells. The plasmids, which were proved in our investigation to contain the secretin gene in the desired orientation, were then screened for the production of secretin. This was shown by radioimmunoassay and gel electrophoretic analysis of the polypeptides that were synthesized in E. coli minicells transformed with the hybrid plasmids. Secretin so produced, whose carboxy terminal residue may not be amidated in contrast with natural porcine secretin, showed the same activity in stimulating pancreatic secretion in a bioassay with anesthetized rats as does natural secretin.

A synthetic gene for human epidermal growth factor (hEGF) was joined to a sequence encoding the signal peptide of Escherichia coli alkaline phosphatase. This hybrid gene was placed under the control of the alkaline phosphatase gene (phoA) promoter in a recombinant plasmid, which was used to transfect E. coli. The hybrid protein that was expressed in host cells under conditions of phosphate limitation was processed accurately during the secretion process, and mature hEGF was recovered in the periplasmic fraction. On the other hand, no EGF was detected in the periplasmic space when the synthetic hEGF gene was not accompanied by the phoA signal sequence.

The noncoding control region of the mitochondrial DNA of various gallinaceous birds was studied with regard to its restriction fragment length polymorphism (RFLP) and sequences of the first 400 bases. Tandem duplication of the 60-base unit was established as a trait unique to the genus Gallus, which is shared neither by pheasants nor by quails. Unlike its close ally Gallus varius (green junglefowl), the red junglefowl Gallus gallus is a genetically very diverse species; the 7.0% sequence divergence was seen between those from Thailand (G. g. gallus and G. g. spadiceus) and the other from the Indonesian island of Java (G. g. Bankiva). Furthermore, the divergence increased to 27.83% if each transversion is regarded as an equivalent of 10 transitions. On the other hand, a mere 0.5-3.0% difference (all transitions) separated various domestic breeds of the chicken from two G. g. gallus of Thailand, thus indicating a single domestication event in the area inhabited by this subspecies of the red junglefowl as the origin of all domestic breeds. Only transitions separated six diverse domesticated breeds. Nevertheless, a 2.75% difference was seen between RFLP type I breeds (White Leghorn and Nagoya) and a RFLP type VIII breed (Ayam Pelung). The above data suggested that although the mitochondrion of RFLP type V was the main contributor to domestication, hens of other RFLP types also contributed to this event.

Significant in vivo stimulation of granulopoiesis was induced in mice by the administration of an extract from the urine of patients with aplastic anemia (AA). Sialic acid has been identified as an important molecular component for the in vivo biological activity of this granulopoietic factor, "granulopoietin," which is distinct and different from endotoxin. Urine from patients with AA was successively fractionated by Sephadex G-50 and DEAE-cellulose chromatography. The resultant extract, which we refer to as AA urinary extract, contained approximately equal to 44 international units of erythropoietin per A unit of protein and induced 15,000 colonies of granulocyte/macrophage precursor cells (granulocyte/macrophage colony-forming units, CFU-gm) per A unit of protein with mouse bone marrow. Eight daily intraperitoneal injections of this extract in mice induced a 6.2-fold increase in peripheral blood granulocytes and a 14.6-fold increase of splenic CFU-gm, with concomitant increases in the proliferation rates of CFU-gm in both bone marrow and spleen. Pretreatment of the AA urinary extract with sialidase significantly diminished these granulopoietic effects in vivo (P less than 0.001). In contrast, both extracts (i.e., native AA and sialidase-treated AA urinary extracts) revealed high granulocyte/macrophage colony-stimulating factor activity in vitro when clonal assays were performed with mouse bone marrow. Increased in vivo and in vitro granulopoietic activities were found in the concanavalin A "break-through" fraction, indicating that these activities were due to protein(s) that did not bind to the lectin. These results reveal that this urinary extract from patients with AA is capable of inducing significant granulopoiesis in mice and that sialic acid is an important component in the maintenance of this granulopoietic effect in vivo but not in vitro.

A RNA molecule has been synthesized that is identical in sequence to Escherichia coli tRNAfMet except that it lacks the base modifications present in the E. coli tRNA. This was achieved by enzymatic joining of chemically synthesized oligonucleotides with chain lengths of 3-10 which were synthesized by the phosphodiester or phosphotriester method. First, quarter molecules of tRNA were constructed by joining of chemically synthesized fragments with RNA ligase. The 5'-quarter molecule (bases 1-20) served as an acceptor in joining reactions with the 3',5'-bisphosphorylated donor molecule (bases 21-34). The 5'-half molecule thus obtained was treated with phosphatase and joined to the 3'-half molecule which was prepared by ligation of the other quarter molecules (bases 35-60, acceptor; bases 61-77, donor) followed by 5'-phosphorylation with polynucleotide kinase. The synthetic tRNA was characterized by oligonucleotide pattern and was partially active in aminoacylation with E. coli methionyl-tRNA synthetase.