2008 Vol. 29, No. 4
2008, 29(4): 353-362.
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The full-length of the cDNA sequence of Beijing fatty chicken ADSL gene was investigated by RT-PCR and RACE in this study. The results demonstrated that the complete ADSL cDNA revealed an open reading frame of 1455 nucleotides coding for 485 amino acid residues. The promoter of chicken ADSL cDNA showed typical features of house-keeping genes: no canonical TATA and CAAT boxes, 72.65% GC in 234 bp near the start codon, and there was a C-28T mutation, which caused the site CTCC mutating to the NRF-2 binding site CTTC. The complete open reading frame of the ADSL gene was inserted into expression vector pGEX-4T-1 and the fusion expression vector pGEX-ADSL was constructed. The pGEX-ADSL was transformed into Escherichia coli BL21 (DE3), positive cloning screened, induced and expresses by IPTG. The SDS-PAGE electrophoresis results showed that there were specific bands, about 80.5 kD and an isoelectric point of 6.79, which reached 26.9% of total cell proteins induced in 5hrs, and was an insoluble inclusion body. Under optimal conditions, ADSL was purified by Glutathione Sepharase 4B affinity chromatography, and Western blotting analysis showed that the fused protein was ADSL. This research was a foundation for further research into ADSL’s biological function and its application identification.
A Human-specific Mutation Leads to Reduced Interaction Between miR-124a and One of Its Target Genes, PLOD3
2008, 29(4): 363-367. doi: 10.3724/SP.J.1141.2008.04363
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MicroRNAs (miRNAs）are a class of noncoding RNAs (20-24 nt) that can post-transcriptionally regulate the expression of protein coding genes in metazoans. Among them, miR-124a, which is preferentially expressed in the brain, controls the neuronal differentiation in mammals. As miRNAs recognize sequences in the 3′ untranslated region (UTR) of the target genes, during human origin, mutations located in the 3′UTRs of the target genes may lead to changes in miRNA regulation. Through target gene prediction and comparative sequence analysis in representative mammalian species, we identified a target gene (PLOD3) of miR-124a, which has a human-specific mutation in the 3′UTR target sequence. Using the in vitro reporter gene system, we discovered that the human specific mutation in the target site of PLOD3 leads to reduced interaction between miR-124a and PLOD3. This result implies that sequence changes located in the 3′UTR segment of the target genes may have functional consequence and eventually contribute to the origin and evolution of human cognition.
Construction and Analysis of A Directional Xenopus laevis Embryonic cDNA Library for Yeast Two-hybrid Using Modified Random Primers
2008, 29(4): 368-372. doi: 10.3724/SP.J.1141.2008.04368
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Here we describe a new strategy for cDNA library construction, in which the random primers directing the first strand cDNA synthesis contain additional d (AC) at their 5′-ends, and the linkers which will be added to the double strand cDNA contain d(GTCG) at the 5′-ends. When the linker is added to the 3′-end of the cDNA, a complete SalI site d(GTCGAC) will form at the 3′-end but not at the 5′-end of the cDNA fragment. After SalI digestion, the 3′-cohesive and the pre-existing 5′-EcoRI cohesive ends are used to introduce the double stranded cDNA into the linearized plasmid vectors. The ligation products were used to transform E.coli to produce a cDNA library. Using this method, we constructed a directional Xenopus laevis embryonic cDNA library for yeast two-hybrid. We checked the ratio of empty vectors, the size of inserts and existence of several genes, the results showed that cDNA library construction was successful.
Searching for Protein-coding Genes Using Microsatellites in Common Carp by Comparing to Zebrafish EST Database
2008, 29(4): 373-378. doi: 10.3724/SP.J.1141.2008.04373
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In this study, an in silico approach was utilized to identify homologies existing between common carp microsatellite sequences and GenBank database using Blastn and Blastx searches. About 875 microsatellite sites with flanking sequences over 50 bp of common carp were first compared to the zebrafish EST database. The results showed that 121 homologies were found using Blastn. Subsequent Blastx searches confirmed 94 sites recorded in the protein database. Except for 33 hypothetical proteins and three unknown proteins, seven out of 58 characterized proteins have been mapped to two linkage maps. In addition, two polymorphic STS markers were developed using matched zebrafish EST sequences by PCR-SSCP method, of which one marker HLJZe33 was mapped successfully. This study was a pilot for comparative studies between common carp and zebrafish, and the results demonstrated that more genetic and genomic resources of zebrafish can be used for the genome research of common carp.
Structure of the Mitochondrial DNA Control Region and Population Genetic Diversity Analysis of Anabarilius grahami (Regan)
2008, 29(4): 379-385. doi: 10.3724/SP.J.1141.2008.04379
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The control region (D-loop) of Anabarilius grahami was amplified by PCR amplification with a pair of specific primers. The sequence with the complete nucleotide control region from A. grahami mitochondrial was cloned and directly sequenced. The length of this region (D-loop) contained 930 bp nucleotides and the T, C, A and G contents were 29.8%, 22.5%, 33.0% and 14.7% respectively. The mtDNA control region of A. grahami could be partitioned into three domains, namely, the termination associated sequence domain, the central conserved sequence domain and the conserved sequence block domain. The extended termination associated sequence (ETAS), two conserved blocks (CSB-F, CSB-D) in the central conserved sequence block domain and three conserved sequence blocks (CSB1, CSB2, CSB3) in the conserved sequence block domain were successfully identified and their homology compared with other fish. The genetic diversity analysis of A. grahami from the Endangered Fish Breeding Center (EFBC) of the Kunming Institute of Zoology (KIZ), Mingxing Fish Cave (Jiangchuan), Niumo Village (Jiangchuan) was analyzed. The result indicated a very low genetic divergence between two natural populations, and the genetic diversity of the population from EFBC was much higher; they had recovered better than others.
2008, 29(4): 386-398. doi: 10.3724/SP.J.1141.2008.04386
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bg-CAT is a naturally existing protein complex of non-lens bg-crystallin and trefoil factor, purified from Bombina maxima skin secretions. In HUVECs, bg-CAT can be rapidly endocytosed via intracellular vacuole formation and translocated to the nucleus to regulate cell fuction. In this paper, we found that it contains conserved Walker B motifs (IILYDEPS, residues 6-13) and Walker A motifs (GQSLSGKS, residues 96-103) in the bg-CAT a-subunit sequence. bg-CAT showed potential NTP-binding and weak GTPase/ATPase activities in vitro. Through Western blotting analysis, we found that the a- and b-subunits of bg-CAT participated in a 150 kDa SDS-stable protein complex formation, which also contained positive ubiquitination signals in the bg-CAT treated HUVEC. Furthermore，under confocal microscopy, the immunofluorescence signals of ubiquitin and bg-CAT subunits were co-localized in the vacuoles that were distributed in the cytoplasm and nucleus. In addition, bg-CAT could induce several tumor cell’s detachment and apoptosis, and selectively kill tumor cells. These findings provide a clue to understand the mechanism of bg-CAT endocysis and nuclear transport, and give an insight to investigate the possible occurrence of similar molecule’s cellular functions and action mechanisms of non-lens bg-crystallins and trefoil factors in mammals.
2008, 29(4): 399-404. doi: 10.3724/SP.J.1141.2008.04399
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A 5′-nucleotidase was isolated and purified from the snake venom of T. albolabris using three steps of chromatography including DEAE-SephadexA-25, Sephadex-G-100 and CM-Sephadex C-50. Using SDS-PAGE and HPLC column chromatography the purified 5′-nucleotidase proved to be homogenous. It was a glycoprotein with a molecular weight of 48.03 kDa. The enzymatic activities of the purified 5′-nucleotidase were 330.33 µg Pi/min mg and 123.56 µg Pi/min mg when using AMP（adenosine monophosphate） and ADP（adenosine diphosphate） as substrates, respectively. Metal ions, including Zn2+, Fe3+ and Cu2+, could inhibit 5′-nucleotidase activity, as did EDTA. Its optimum pH was nine and its optimum temperature was 50°C. It has a potent inhibitory effect on rabbit platelet aggregation induced by ADP.
Influence of Different FGF2-expressing Feeder Layers on the Self-renewal and Pluripotency of Rhesus Monkey Embryonic Stem Cells
2008, 29(4): 405-414. doi: 10.3724/SP.J.1141.2008.04405
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Transgene and RNAi were used to establish five different FGF2-expressing monkey ear skin fibroblast (MESF) cell lines, the FGF2 over-expressed line (f1), the negative control of f1 (f2), RNA interfered line (f3), the negative control of f3 (f4) and the non-treated control (f5). The expression ratio of FGF2 in these lines was f1: f2: f3: f4: f5=4∶2∶1∶2∶2. The results indicated that c-fos, TGF-β1, INHBA, Gremlin1 were upregulated in f1 but downregulated in f3, while BMP4, TGF-β2 were downregulated in f1 but upregulated in f3, which implied that endogenous FGF2 affected the TGF-β signaling pathway and the expression level of related genes changed in MESFs. Further analysis of rhesus monkey embryonic stem cells (RhESCs) supported by these MESFs showed that RhESCs on f1 grew more quickly than those in other groups, in which the expression level of c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog and Sox2 was higher, while that of BMP4 was lower; in contrast, RhESCs on f3 grew more slowly than in other groups, in which the expression level of c-fos，TGF-β1，INHBA，Gremlin1，OCT-4，Nanog and Sox2 declined, while that of BMP4 and TGF-β2 increased. EBs from these RhESCs expressed early markers representing all germ layers, but the expression levels of markers in RhESCs on f1 were lower than in other groups. These findings demonstrated that different FGF2 expression in feeder layers can not only influence expression of relative genes in MESFs, but also affect proliferation and self-renewal of ESCs.
2008, 29(4): 415-420. doi: 10.3724/SP.J.1141.2008.04415
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A “2＋2 days” fast protocol for the generation of dendritic cells（DC） from high purity human monocytes in vitro has been established. During the 2-step differentiation and activation process, we demonstrated that 2 days of culture with GM-CSF and IL-4 were sufficient to generate immature DCs capable of antigen uptake. Similarly the mature DCs were activated with a cocktail of rTNF-α, IL-1β, IL-6, and PGE2 from immature DC in 2 days. This “2＋2” fast DC had the same phenotype and function as the “6＋2” standard DC.
Establishment and Characterization of THP-1 Cell Model Stably Expressing HIV-1 Auxiliary Protein Rev
2008, 29(4): 421-426. doi: 10.3724/SP.J.1141.2008.04421
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HIV-1 auxiliary proteins play a critical role in both the infection of HIV-1 target cells and the morbidity of AIDS. Rev, as one of the HIV-1 auxiliary proteins, is essential for the replication of HIV-1. As a membrane-shuttle protein, it can regulate the transportation of HIV-1 structural protein’s mRNA. The rev gene from HIV-1 was transfected into THP-1 cells. The Rev-stably-expressed cell model was generated by electroporation and then sorted and selected with G418. The cell model was characterized at both mRNA and protein levels with an RT-PCR assay, fluorescent observation, and flow cytometry analysis. The rev gene was successfully transfected into THP-1 cells and stably expressed. This is a useful result for future research of the interaction between Rev and cells.
2008, 29(4): 427-430. doi: 10.3724/SP.J.1141.2008.04427
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In the present study, the effects of fluorocitrate on the proliferation of cultured glioblastomas G422 cells were investigated. Proliferation of glioblastomas G422 cells was measured by MTT assay at 36 h, 48 h and 60 h after fluorocitrate (0.0025 mmol/L, 0.005 mmol/L, 0.01 mmol/L, 0.025 mmol/L and 0.1 mmol/L) treatment. Our results showed that: (1) Fluorocitrate inhibited the proliferation of glioblastomas G422 cells in a dose dependent manner; (2) The inhibition rate increased with treatment time at high concentrations of fluorocitrate (0.01 mmol/L, 0.025 mmol/L and 0.1 mmol/L); (3) The inhibition rate did not increase with treatment time at low concentrations of fluorocitrate (0.0025 mmol/L and 0.005 mmol/L). This study indicated that fluorocitrate inhibited the proliferation of glioma cells as a function of fluorocitrate concentration and treatment time.
Distribution of Like-muscarinic Acetylcholine Receptor M2 in the Brain of Three Castes of Polyrhachis vicina
2008, 29(4): 431-437. doi: 10.3724/SP.J.1141.2008.04431
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The cholinergic system plays an important role in the central nervous system of insects and is closely related to the complex behavior of insects. The immunohistochemical technique was performed to detect the expression of like-muscarinic acetylcholine receptor M
2 in the brain of three castes of Polyrhachis vicina. A positive expression of like-muscarinic acetylcholine receptor M2 was observed in the mushroom body, central body and antennal lobes of the ant brain; but there is great diversity in their location and intensity among worker, queen and male ants. It is speculated that like-muscarinic acetylcholine receptor M2 plays a critical role in the central nervous system, in terms of projecting visual information and olfactory information into the protocerebrum and integrating many inputs.
Faunistic and Distribution of Eumolpidae (Coleoptera: Chrysomeloidea) Species in Heilongjiang Province, China
2008, 29(4): 438-446. doi: 10.3724/SP.J.1141.2008.04438
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Surveys were conducted to study faunistic and distribution of Eumolpidae species in Heilongjiang Province between April 2005 and October 2007. Fauna of Eumolpidae in Heilongjiang Province were first divided into 11 geographic sub-regions based on the principles of zoogeographic distribution in China and using distribution records of Eumolpidae species in 11 geographic sub-regions, a C matrix was constructed then a clustering analysis maximum value method was used. A total of 57 species (subspecies) belonging to 14 genera were recorded, including, Cryptocephalinae, Clytrinae, and Eumolpinae, 34 species (accounting for 59.6% of the total), 15 species (26.3%), and 8 species (14.1%) respectively. Species of Lamprosomatinae and Chlamisinae have not been found in Heilongjiang Province. Only nine species (belonging to three genera) are distributed in the Palearctic Region and one species is endemic to Heilongjiang Province. The others are widely distributed in the Palearctic and Oriental Region. The analysis indicated that 71.9% of the total species (41 species) are distributed in the southeast of the Province, where the climate is warmer and more humid than other regions. Fifteen and 22 species found in Xing’an Mountain and Songnen Plain, account for 26.3% and 38.6% of the total number of species respectively. The clustering analysis of region similarity of Eumolpidae demonstrated that the closest relationship was between the western and eastern parts of Mudanjiang Region, then Songjiang Plain Region and Northern Songnen Region. The western dry grasslands region had the most alienated relationship with the others.
Echolocation Call, Roost and ND 1 Sequence Analysis of New Record of Nyctalus plancyi（Chiroptera: Vespertilionidae）on Hainan Island
2008, 29(4): 447-451. doi: 10.3724/SP.J.1141.2008.04447
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One male bat was captured by mist-net on a telegraph pole in the Li Autonomous County, Hainan Island in November 2007. It was identified as Nyctalus plancyi （Chiroptera: Vespertilionidae） based on the morphological data and mitochondrial ND1 gene sequence, and presented as the first record from Hainan Island. N. plancyi, which roosted daily on a telegraph pole in Hainan Island, emitted a pulse with an energy frequency between 33 - 34 kHz, and pulse duration between 1.3-1.9 ms, maximum identity based on ND1 sequence analysis between Hainan Island and Sichuan was 99%.
2008, 29(4): 452-454. doi: 10.3724/SP.J.1141.2008.04452
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A new species of the genus Protocobitis, was discovered in an underground water source (110 m depth) located about 5 km away from the county town of Wuming, Guangxi, China, in May 2006. The new species, Protocobitis polylepis sp. nov., is distinguished from the single congener by the following characteristics: 1) light black pigmentation present vs. no pigmentation; 2) entire body covered with scales except for its head and abdomen vs. rudimentary scales present only along the midline of the body; 3) head length 24.1%-24.8% of SL vs. 19.8-22.1% of SL; 4) body depth 16.2%-16.3% of SL vs. 11.5%-13.0% of SL; 5) Inner rostral barbel length 19.8%-21.0% of head length vs. 9.4%-11.8% of head length; outer rostral barbel length 28.6%-30.2 % of head length vs. 15.3%-21.8% of head length; maxillary barbell length 44.6%-46.0% of head length vs. 22.4%-31.8% of head length.
One new species of the genus Tetrix Latreille, Tetrix xiangzhouensis sp. nov. is described, and the male of Bolivaritettix luochengensis Deng, Zheng et Wei is reported for the first time. New species is similar to Tetri fuchuanensis Zheng，1998, but differs in a number of ways. Width of vertex equal to or slightly narrower than width of an eye, anterior margin of vertex not protruding beyond eyes, length of a segment in middle of antenna about 7-8 times longer than width, lateral keels of prozona constricted backward, hind process of pronotum reaching two thirds of hind tibia, lower margins of anterior and middle femora ornate with sparse hairs.
2008, 29(4): 459-464. doi: 10.3724/SP.J.1141.2008.04459
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The metabolism of organisms is carried out through a series of metabolic pathways. How these ingeniously regulated and collaborated metabolic pathways evolved has been a fascinating and important question for some time. Since the ‘retro-evolution hypothesis’ was first put forward in 1945, there have been seven main hypotheses concerning the evolution of metabolic pathways, including ‘retro-evolution of pathways, ‘semi-enzymatic theory’, ‘forward development model’, ‘enzyme recruitment hypothesis’, ‘specialization of multifunctional enzyme’, ‘pathway duplication’, and ‘de novo invention of pathways’. Among them, the most popular hypotheses are ‘retro-evolution’ and ‘enzyme recruitment’, and recently greater attention has been paid to the ‘specialization of multifunctional enzyme’ hypothesis, due to its strong theoretical basis and experimental evidence. Here, all of the seven hypotheses were reviewed, and the development prospect of this field analyzed.