Molecular cloning, overexpression, purification, and sequence analysis of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide.

The complementary DNA (cDNA) of the giant panda (Ailuropoda melanoleuca) ferritin light polypeptide (FTL) gene was successfully cloned using reverse transcription-polymerase chain reaction technology. We constructed a recombinant expression vector containing FTL cDNA and overexpressed it in Escherichia coli using pET28a plasmids. The expressed protein was then purified by nickel chelate affinity chromatography. The cloned cDNA fragment was 580 bp long and contained an open reading frame of 525 bp. The deduced protein sequence was composed of 175 amino acids and had an estimated molecular weight of 19.90 kDa, with an isoelectric point of 5.53. Topology prediction revealed one N-glycosylation site, two casein kinase II phosphorylation sites, one N-myristoylation site, two protein kinase C phosphorylation sites, and one cell attachment sequence. Alignment indicated that the nucleotide and deduced amino acid sequences are highly conserved across several mammals, including Homo sapiens, Cavia porcellus, Equus caballus, and Felis catus, among others. The FTL gene was readily expressed in E. coli, which gave rise to the accumulation of a polypeptide of the expected size (25.50 kDa, including an N-terminal polyhistidine tag).

Comparative analysis and molecular characterization of genomic sequences and proteins of FABP4 and FABP5 from the giant panda (Ailuropoda melanoleuca).

Fatty acid binding proteins (FABPs) are a family of small, highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. In this study, cDNA and genomic sequences of FABP4 and FABP5 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using reverse transcription polymerase chain reaction (RT-PCR) technology and touchdown-PCR. The cDNAs of FABP4 and FABP5 cloned from the giant panda were 400 and 413 bp in length, containing an open reading frame of 399 and 408 bp, encoding 132 and 135 amino acids, respectively. The genomic sequences of FABP4 and FABP5 were 3976 and 3962 bp, respectively, which each contained four exons and three introns. Sequence alignment indicated a high degree of homology with reported FABP sequences of other mammals at both the amino acid and DNA levels. Topology prediction revealed seven protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, two N-myristoylation sites, and one cytosolic fatty acid-binding protein signature in the FABP4 protein, and three N-glycosylation sites, three protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, one N-myristoylation site, one amidation site, and one cytosolic fatty acid-binding protein signature in the FABP5 protein. The FABP4 and FABP5 genes were overexpressed in Escherichia coli BL21 and they produced the expected 16.8- and 17.0-kDa polypeptides. The results obtained in this study provide information for further in-depth research of this system, which has great value of both theoretical and practical significance.

cDNA, genomic sequence cloning and overexpression of giant panda (Ailuropoda melanoleuca) mitochondrial ATP synthase ATP5G1.

The ATP5G1 gene is one of the three genes that encode mitochondrial ATP synthase subunit c of the proton channel. We cloned the cDNA and determined the genomic sequence of the ATP5G1 gene from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively. The cloned cDNA fragment contains an open reading frame of 411 bp encoding 136 amino acids; the length of the genomic sequence is of 1838 bp, containing three exons and two introns. Alignment analysis revealed that the nucleotide sequence and the deduced protein sequence are highly conserved compared to Homo sapiens, Mus musculus, Rattus norvegicus, Bos taurus, and Sus scrofa. The homologies for nucleotide sequences of the giant panda ATP5G1 to those of these species are 93.92, 92.21, 92.46, 93.67, and 92.46%, respectively, and the homologies for amino acid sequences are 90.44, 95.59, 93.38, 94.12, and 91.91%, respectively. Topology prediction showed that there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, five N-myristoylation sites, and one ATP synthase c subunit signature in the ATP5G1 protein of the giant panda. The cDNA of ATP5G1 was transfected into Escherichia coli, and the ATP5G1 fused with the N-terminally GST-tagged protein gave rise to accumulation of an expected 40-kDa polypeptide, which had the characteristics of the predicted protein.

cDNA, genomic sequence cloning and overexpression of ribosomal protein gene L9 (rpL9) of the giant panda (Ailuropoda melanoleuca).

The ribosomal protein L9 (RPL9), a component of the large subunit of the ribosome, has an unusual structure, comprising two compact globular domains connected by an α-helix; it interacts with 23 S rRNA. To obtain information about rpL9 of Ailuropoda melanoleuca (the giant panda) we designed primers based on the known mammalian nucleotide sequence. RT-PCR and PCR strategies were employed to isolate cDNA and the rpL9 gene from A. melanoleuca; these were sequenced and analyzed. We overexpressed cDNA of the rpL9 gene in Escherichia coli BL21. The cloned cDNA fragment was 627 bp in length, containing an open reading frame of 579 bp. The deduced protein is composed of 192 amino acids, with an estimated molecular mass of 21.86 kDa and an isoelectric point of 10.36. The length of the genomic sequence is 3807 bp, including six exons and five introns. Based on alignment analysis, rpL9 has high similarity among species; we found 85% agreement of DNA and amino acid sequences with the other species that have been analyzed. Based on topology predictions, there are two N-glycosylation sites, five protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, two tyrosine kinase phosphorylation sites, three N-myristoylation sites, one amidation site, and one ribosomal protein L6 signature 2 in the L9 protein of A. melanoleuca. The rpL9 gene can be readily expressed in E. coli; it fuses with the N-terminal GST-tagged protein, giving rise to the accumulation of an expected 26.51-kDa polypeptide, which is in good agreement with the predicted molecular weight. This expression product could be used for purification and further study of its function.

cDNA, genomic sequence cloning and overexpression of the ribosomal protein S13 gene in the giant panda (Ailuropoda melanoleuca).

The cDNA and the genomic sequence of ribosomal protein S13 (RPS13) of the giant panda (Ailuropoda melanoleuca) was cloned using reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR, respectively. These two sequences were sequenced and analyzed, and the cDNA of the RPS13 gene was overexpressed in Escherichia coli BL21. We compared the nucleotide sequences of the coding region and the amino acid sequences with those of seven other mammalian species retrieved from GenBank. The cDNA fragment of the RPS13 cloned from the giant panda is 496 bp in size, containing an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 2277 bp, with five exons and four introns. The coding sequence shows a high degree of homology to those of Homo sapiens, Bos taurus, Canis lupus familiaris, Macaca mulatta, Mus musculus, Rattus norvegicus, and Pan troglodytes; the degree of homology was 91.23, 94.30, 94.74, 92.11, 87.94, 87.72, and 91.45%, respectively. The homologies for the deduced amino acid sequences reached as high as 99%. Primary structure analysis revealed that the molecular weight of the putative RPS13 protein is 17.22325 kDa, with a theoretical pI of 10.42. Based on topology prediction, there is one protein kinase C phosphorylation site, one casein kinase II phosphorylation site, two N-myristoylation sites, and one ribosomal protein S15 signature in the RPS13 protein of the giant panda. The RPS13 gene can be expressed in E. coli and the RPS13 protein fused with the N-terminally GST-tagged form, which gave rise to the addition of an expected 43-kDa polypeptide.

Giant panda ribosomal protein S14: cDNA, genomic sequence cloning, sequence analysis, and overexpression.

RPS14 is a component of the 40S ribosomal subunit encoded by the RPS14 gene and is required for its maturation. The cDNA and the genomic sequence of RPS14 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively; they were both sequenced and analyzed. The length of the cloned cDNA fragment was 492 bp; it contained an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 3421 bp; it contains four exons and three introns. Alignment analysis indicates that the nucleotide sequence shares a high degree of homology with those of Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, and Danio rerio (93.64, 83.37, 92.54, 91.89, 87.28, 84.21, and 84.87%, respectively). Comparison of the deduced amino acid sequences of the giant panda with those of these other species revealed that the RPS14 of giant panda is highly homologous with those of B. taurus, R. norvegicus and D. rerio (85.99, 99.34 and 99.34%, respectively), and is 100% identical with the others. This degree of conservation of RPS14 suggests evolutionary selection. Topology prediction shows that there are two N-glycosylation sites, three protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, four N-myristoylation sites, two amidation sites, and one ribosomal protein S11 signature in the RPS14 protein of the giant panda. The RPS14 gene can be readily expressed in Escherichia coli. When it was fused with the N-terminally His-tagged protein, it gave rise to accumulation of an expected 22-kDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained can be purified for studies of its function.

[An analysis of population growth in Romania].

PIP: After liberation in Romania the high birth rate, high mortality rate, and high population growth rate pattern changed to a low birth rate, low mortality rate, and low growth rate pattern. Higher standard of living and educational level, the increasing involvement of women in social and economic activities, the rapid development of cities, and the lower infant mortality rate are the 4 main factors responsible for this development. Consequently, Romania is facing a problem of increasing labor shortages. People who would otherwise be in the labor force are in school to allow the rapid development in science and technology. The increasing proportion of older retirees in the population also decreases the labor supply. Agricultural mechanization has provided labor to support industrialization in the past. Future increases will emphasize irrigation and soil improvement rather than mechanization. The Romanian government has established 6 new laws to stimulate population growth. First, award bonuses to families with more children and tax childless couples. Second, eliminate factors destabilizing families, preference to young couples, and restrict abortion and divorce. Third, protect women's societal rights through bonuses to mothers and paid maternity leave to pregnant women. Fourth, reduce mortality rate and improve people's health and life span. Fifth, better utilize the labor force and redistribute the population by economic methods. Six, use longterm procedures to achieve the best population structure and to have a younger population.^ieng