Protodioscin-glycosidase-1 hydrolyzing 26-O-ß-D-glucoside and 3-O-(1 → 4)-α-L-rhamnoside of steroidal saponins from Aspergillus oryzae.

A novel protodioscin-(steroidal saponin)-glycoside hydrolase, named protodioscin-glycosidase-1 (PGase-1), was purified and characterized from the Aspergillus oryzae strain. The molecular mass of this enzyme was determined to be about 55 kDa based on SDS-polyacrylamide gel electrophoresis. PGase-1 was able to hydrolyze the terminal 26-O-ß-D-glucopyranoside of protodioscin (furostanoside) to produce dioscin (spirostanoside), and then further hydrolyze the terminal 3-O-(1 → 4)-α-L-rhamnopyranoside of dioscin to form progenin III. However, PGase-1 could hardly hydrolyze the 3-O-(1 → 2)-α-L-rhamnopyranoside of progenin III, 3-O-ß-D-glucoside of trillin, and the 1-O-glycosides of ophiopogonin D (steroidal saponin). In addition, PGase-1 also could hydrolyze the α-D-galactopyranoside, ß-D-glucopyranoside, and ß-D-galactopyranoside of p-nitrophenyl-glycosides, but the enzyme could not hydrolyze the α-D-mannopyranoside, α-L-arabinopyranoside, α-D-glucopyranoside, ß-D-xylopyranoside, and α-L-rhamnopyranoside of p-nitrophenyl-glycosides. These new properties of PGase-1 were significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB. The gene (termed as pgase-1) encoding PGase-1 was cloned, sequenced, and expressed in Pichia pastoris GS115. The complete nucleotide sequence of pgase-1 consists of 1,725 bp. The recombinant PGase-1 from recombinant P. pastoris GS115 strain also showed the activity hydrolyzing glycosides of steroidal saponins which was similar to that of the wild-type PGase-1 from A. oryzae. The PGase-1 gene is highly similar to Aspergilli α-amylase (EC 3.2.1.1), and PGase-1 should be classified as glycoside hydrolase family 13 by the method of gene sequence-based classification. But the enzyme properties of PGase-1 are different from those of α-amylase in this family.

Aspergillus niger DLFCC-90 rhamnoside hydrolase, a new type of flavonoid glycoside hydrolase.

A novel rutin-α-L-rhamnosidase hydrolyzing α-L-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the α-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13.

[Progress of the PCR amplification techniques for chromosome walking].

Various PCR-based methods are available for chromosome walking from a known sequence to an unknown region. It is promising in genome-related research for the following experiments: promoter cloning, obtaining non-conservative parts of genes in new species, identification of T-DNA or transposon insertion sites and filling in gaps or unknown chromosome regions in genome sequencing. These methods consisted of three types: inverse PCR, ligation-mediated PCR and specific primer PCR. In this review, we illustrated and compared the current techniques.

The complete mitochondrial genomes sequences of Asio flammeus and Asio otus and comparative analysis.

The complete mitochondrial genomes of Asio flammeus and Asio otus were sequenced and found to span 18858 bp and 18493 bp, respectively. It is surprising to find the former to be the largest among all avian mitochondrial genomes sequenced so far. The two genomes have very similar gene order with that of Gallus gallus, neither contains the pseudo control region, but both have a single extra base, namely Cytidine, at position 174 in ND3 gene. The control regions of Asio flammeus and Asio otus' mitochondrial genomes span 3288 bp and 2926 bp respectively, which are the longest among vertebrates except for Myxine glutinosa and contribute to the large size of two genomes. The 3' end of the control region of Asio flammeus and Asio otus contains many tandemly repeated sequences, which are highly similar to a putative control element, i.e. Mt5, and may form stable stem-loop secondary structures. Such repeated sequences probably play an important role in regulating transcription and replication of mitochondrial genome. Our results may provide important clues for uncovering the origin and evolution mechanisms of mitochondrion genome.

[Determination of transcription initiation site of chicken ovalbumin gene and construction of its expression vector].

To determine the core promoter of chicken ovalbumin gene and 5' upstream regions, the transcription initiation site of ovalbumin gene was confirmed by 5'RACE method, at the same time, the regulatory elements of chicken ovalbumin gene were determined by sequence analysis. To investigate the ability of regulatory elements to direct the exogenous gene expression, 1.5 kb fragment and 2.9 kb fragment were amplified by PCR method. Two fragments were subcloned to mammalian expression vector pGFP-N2 by recombinant DNA technology, the CMV promoter was cut off from pGFP-N2. Two expression vectors were constructed, one is the P2.9koval-GFP including promoter,first exon,first intron of chicken ovalbumin gene, and the other is the P1.5koval-GFP including first intron of chicken ovalbumin gene. Restriction enzyme digestion and DNA sequence analysis revealed that 5' upstream regions of ovalbumin gene were not only identical to those of the published chicken ovalbumin gene, but also were contained in the recombinant vector.

[Compariative study of mitochondrial tRNA gene sequence and secondary structure among fifteen Predatory birds].

Three major clusters of mitochondrial tRNA genes (tRNA(Ile) -tRNA(Gln) -tRNA(Met), tRNA(Trp)- tRNA(Ala) -tRNA(Asn)- tRNA(CYs) -tRNA(Tyr) and tRNA(His) tRNA(Ser)(AGY) -tRNA(Leu)(CUN) from 13 species of Predatory birds were amplified and sequenced. The length of these tRNA clusters was similar among species (212 approximately 214 bp, 353 approximately 362 bp, 205 approximately 208 bp, respectively), and 47% of the sequences were variable, 67% of which were involved in the loop regions. The stem regions were relatively conserved, and the variable base pairs were under the restriction of compensatory changes or G-U wobble pairing which could be regarded as mechanisms for maintaining a stable secondary structure. Maximum-parsimony (MP) and Neighbor joining (NJ) phylogenetic trees were constructed using all the tRNA gene sequences or stein-forming nucleotides with Caprimulgus indicus as outgroup. We found that the bootstrap values for branches of trees using the tRNA sequences were commonly higher than the others, therefore the phylogenetic relationship of Predatory birds reflected by these data may be closer to the truth. Phylogenetic analyses indicated that Accipitridae was closer to Strigidae instead of Falconidae, and the classification of Tytonidae was different from the conclusion from the previously morphological and DNA-DNA hybridization studies. By comparing the secondary structure among taxa we found that the characters of nucleotide insertions and deletions in some tRNA genes have synapomorphies, suggesting that these characters may be useful for resolving the phylogenetic relationship of different families in Predatory birds with higher phylogenetic performance.

[Sequence variation of mitochondrial cytochrome b gene and phylogenetic relationships among twelve species of Charadriiformes].

Studies of the phylogenetic relationships of the Charadriiformes have been largely based on conservative morphological characters. During the past 10 years, many studies on the evolutionary biology of birds adopted phylogenetic information obtained from mitochondrial DNA, but few work on the Charadriiformes has been reported to date. Therefore, phylogenetic relationships and classification of the Charadriiformes remains controversial. In this study, we try to shed light on these relationships via DNA sequence analysis of the mitochondrial Cyt b gene in 12 species of Charadriiformes. It was a preliminary study of the origin and evolution of the species by using nucleotide sequence data. Using the well-known PCR techniques, the complete mitochondrial Cyt b gene sequences were amplified and sequenced respectively from Charadrius mongolus, Charadrius alexandrinus, Numenius madagascariensis, Numenius arquat, Numenius phaeopus, Tringa totanus, Tringa glareola, Xenus cineres, Arenaria interpres, Calidris tenuirostris, Recurvirostra avosetts and Haematopus ostralensis. The 1143 bp long DNA sequences of the gene from these species were obtained, in which 381 variable sites were identified without insertions or deletions. The nucleic acid sequence variation of the mitochondrial Cyt b gene was 5.16%-16.01% among these species. Phylogenetic trees constructed using the NJ method, MP method and ML method with Ciconia ciconia as the outgroup indicate that the 12 species of Charadriiformes examined in this study are clustered in two major clades. The first clade includes T. totanus, T. glareola, A. interpres, C. tenuirostris, X. cineres, N. madagascariensis, N. arquata and N. phaeopus. The second one includes C. mongolus, C. alexandrinus, R. avosetts and H. ostralensis. Our molecular data show that the phylogenetic relationships among species of Scolopacidae are consistent with the classification based on morphological studies; R. avosetts and H. ostralensis are relatively closer, and form a sister group, and then form paraphyletic group with a sister group which comprised of C. mongolus and C. alexandrinus. The results support Sibley's opinion of assigning R. avosetts and H. ostralensis which form Recurvirostrinae as a taxon of the Charadriidae, and the Charadriidae dividing into two subfamilies: Recurvirostrinae and Charadriinae respectively.

[A high-efficiency method of sorting and sequencing of functional genes].

The implementation of the Human Genome Project preludes the analyzing of biologic genomes. Following the successful analysis of diverse biologic genomes, it becomes more and more important to research the functions of genes and to find new functional genes. In this article, we use the techniques of Megaclone, Megasort and MPSS to sort and sequence effectively different functional genes.

[A review on mitochondrial DNA of avian].

Mitochondrial DNA as a genetic marker has been successfully applied to the study of molecular evolution of birds. The apparently maternal inheritance of mitochondrial DNA and its fast evolution in primary sequence has made it attractive in population and evolutionary genetics. Mitochondrial DNA of birds displays two characteristics not seen in other vertebrates mtDNA, that is, a novel gene order and the absence of an equivalent to the light-strand replication origin. The research on polymorphism of mtDNA can resolve phylogenies of birds both at lower and higher taxonomic levels. Here we review progress on avian molecular evolution in recent years,and make preliminary studies of the development in this field.

[Studies of the clone and cell expression of human lactoferritin gene].

In this paper,we directly cloned 2.366 Kb cDNA sequence of human lactoferrin gene and 800 bp 5'flank regulatory sequence of beta/lactoglobulin gene from goat by PCR,then connected them with the expression vector pLNCX. The recombinant plasmid pLNCXHLF containing human lactoferrin gene cDNA was transfected into mice mammary tumor cell line MA/782 after liposome transinfection. Positive single clone cells were selected with G418 and by PCR. After proliferating,the transfected cells immobilized and cultured in sodium alginate were induced by hormone. The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLF protein, whose MW is 34 KD. The highest amount detected by ELISA reached 65 mg/l medium/10(5) cells. The result of antibacterial experiment indicates that the recombinant hLF protein has the effect of inhibiting E.coli proliferation;moreover,its activity is superior to the commercial available hLF's.