Studies on resistance characteristic and cDNA sequence conservation of transferrin from crucian carp, Carassius auratus.

Transferrin (Tf) is a kind of non-heme beta-globulin with two iron ions (Fe(3+))-binding sites. To prove Tf's physiological functions, Fe(3+)-proteins, serum iron contents, and total iron-binding capabilities were tested for Tfs of crucian carps (Carassius auratus) and sliver carps (Hypophthalmichthys molitrix). The above results demonstrated that sliver carps shared 1/3 Tf alleles with crucian carps; Tf of crucian carps had stronger Fe(3+)-binding ability and transportation ability in plasma than that of sliver carps. In addition, the results of oxygen consumption experiments indicated that crucian carps had the higher oxygen utility rate than sliver carps. For acute hypoxia exposure assay, normoxic gas mixture, hypoxic gas mixture A, and hypoxic gas mixture B were used to induce oxygen-regulated gene expression of crucian carps in acute hypoxia. The results of quantitative real-time PCR revealed that mRNA levels of Tf gene, Tfr gene and ATPase gene were down-regulated in acute hypoxia but mRNA level of LDHa gene was up-regulated in acute hypoxia. The results of crucian carp Tf-cDNA sequence analysis showed that cDNA regions of two Fe(3+)-binding sites were T(747)-T(1026) and T(1737)-A(1884) based on the principle of bioinformatics. The sequence conservation of two Fe(3+)-binding sites was higher than that of the other five regions, which were confirmed according to the subregion model of Tf-cDNA sequence.

Isolation and chromosomal localization of ZFX homologue in genome of rice field eel (Monopterus albus Zuiew).

When used as a probe in rice field eel (Monopterus albus Zuiew) genomic Southern blotting hybridization, the giant panda Zfx gene hybridized strongly to a fragment of about 9.5 kb. A 512 bp long DNA fragment has been isolated by polymerase chain reaction from rice field eel genomic DNA using the primers for amplifying zinc finger repeats 7 to 13 of mammalian and reptilian ZFX-related genes. Cloned in pBS, four recombinant plasmids were selected randomly from male and female specimens and sequenced. The nucleotide sequences in these clones were identical and showed 88% and 87% identity to human ZFX and ZFY respectively. But its extent of homology was greater with American alligator Zfc (90%). And the amino acid sequences of the putative protein showed 95.9%, 95.9% and 93.5% identity to human ZFX and ZFY and American alligator Zfc respectively. Thus, the cloned sequence encodes a homologue of mammalian ZFX/ZFY and was named Zfa for rice field eel zinc finger domain gene. It appears that the mammalian and reptilian ZFX-related genes evolved from fish ancestors with a considerable degree of conservation. By fluorescence in situ hybridization, the Zfa has been mapped to rice field eel chromosome 1 and at the position of 60.1 +/- 0.38 from the centromere. Chromosomal mapping of fish genes related to mammalian X-linked genes might lead to further understanding of the evolution of vertebrate sex chromosomes.

[Chromosomal localization of the hormone-sensitive lipase gene (Hsl) in rice field eel].

Adipose tissue triacylglycerols are the quantitatively most important source of stored energy in animals. Hormone-sensitive lipase encoded by hormone-sensitive lipase gene (Hsl) is a multifunctional enzyme that catalyzes the hydrolysis of triacylglycerol stored in adipose tissue and cholesterol esters in the adrenals, ovaries, testes and macrophages. Using pig Hsl gene inserted into pBS labeled by the radioactive isotope and the digoxigenin as the probes respectively one band, 11.5kb, has been shown to hybridized with total DNA of rice field eel digested with Pst I by Southern blotting and Hsl gene has been assigned to metaphase chromosome 5, at the position of 78.35+/-1.26 from the centromere in rice field eel by fluorescent in situ hybridization (FISH). The mapping results are corresponding to that of "specific-chromosomal DNA pool" obtained by chromosome microisolation used to map gene and the mapping result is more accurate. The results of the study further illustrate the importance of the presence of Hsl gene in rice field eel genome and provide the first FISH mapping data for rice field eel chromosome 5. The current studies would advance the addition of known genetic markers and the construction of high resolution genetic map in rice field eel genome.

[Construction and homologous detection of a DNA plasmid library specific for Z chromosome of quail].

A simple method was used to adapt a standard light microscope for the collection of quail Z chromosomes from mitotic-metaphase spreads. The microisolated chromosomes were subjected to proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed PCR (DOP-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis, and smears of DNA revealed that ranged in size from 200-1 400 bp, without any evidence of preferential amplification. The second-round PCR products were cloned into pBluescript plasmids to construct a Z chromosome-specific DNA library. The size range of the cloned inserts was 200-1 400 bp. Using inserted fragments from the library as probes, chromosome painting was performed on quail chromosomes. The results showed that Z chromosomes of quail were completely covered by strong signals and there were little signals on other chromosomes. It was indicated that inserted DNA of the library was specific to the Z chromosome of quail. The library can be used as chromosome painting probe to detect conserved syntenic groups on the chromosomes of other related species and study mechanisms of sex-chromosomes evolution in birds.

[Chromosomal localization of rice field eel Hox genes by PRINS].

The genome duplication and chromosome rearrangement are two kinds of evolution models at the chromosome level during the evolution of vertebrate genome. And Hox genes are the powerful proves to support the evolution theory of genome duplication, which has been found recently. In this study, the chromosomal localization of rice field eel Hox genes has been carried out by PRINS. The mapping results indicated that 6 Hox clusters might exist in rice field eel genome, and these clusters were localized on chromosome 1, 2, 3, 6, 8, 10 and at the position of 28.24 +/- 2.88, 4.55 +/- 1.39, 13.89 +/- 2.03, 74.32 +/- 1.86, 38.03 +/- 2.41, 58.18 +/- 2.05 from the centromere respectively. The mapping results that Hox genes were localized on chromosome 1, 3, 6 and 10 in the study are corresponding to that by chromosome microdissection. The chromosomal localization of rice field eel Hox genes will help us to discover the origin and evolution of rice field eel chromosomes, and provide cellular genetic proves of this special species to support the evolution theory of genome duplication.

[Explore on the advanced method of G-banding of Rana plancyt].

Using the technique of natural aging,chloroform and EDTA treatment,well-resolved G-banding patterns were successfully obtained in Rana plancyt's mitosis chromosome firstly. The G-banding patterns appeared distinctive. When analyzing some homologous chromosomes from the same metaphase figures and four macrochromosomes from different metaphase figures,the characteristic and the number of the bands were well matched. The method in this study is also economic and simple and the result is stable and reliable. The possible mechanism of G-banding was primarily discussed. The application of this method in the cytogenetics research of Rana plancyt and other amphibians is expected.