Teaching practice in human genetics integrated into general education.

General education is an important part in higher education, which emphasizes the educational idea of integration of generality with specialty, and practices people-oriented education concept. However, there are some difficulties and puzzles in general education. Now the general education system with Chinese characteristics is needed to be established through practice and development. In this paper, we enumerate how to integrate knowledge of human genetics in practice of general education, teaching cases, and relevant analysis with concepts of general education. Using questions as "what are human beings?" as a leverage, we introduce teaching contents closely related to daily life. For example, we explain the past, present and future of human beings through contemporary evolutionary genomics teaching. In addition, we introduce problem-based deep thinking for students, thus integrating classical attributes of human beings into general education.

[Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway].

All proteins in eukaryotic cells are continually being degraded and replaced. Autophagy and the ubiquitin-proteasome system are two mechanisms for intracellular protein degradation. Autophagy is mediated by lysosome, and is further divided into chaperone-mediated autophagy, microautophagy and macroautophagy. The ubiquitin-proteasome system is highly complex and mediated by ubiquitin, which participates in intracellular protein degradation in a specific manner. It is now known that degradation of intracellular proteins is involved in regulation of a series of cellular processes, including cell-cycle division, DNA repair, cell growth and differentiation, quality control, pathogen infection, and apoptosis. The aberrations in the protein degradation systems are involved in many serious human diseases. The present review summarizes the mechanisms of protein degradation and related human diseases.

[Alternative development of early embryogenesis: sex determination].

Sex determination is a complex regulatory process of early embryogenesis. Embryo must make a developmental decision to develop as a male or female during gonadogenesis. This paper reviews genetic systems of sex determination, gonadogenesis, key genes involved in sex determination of vertebrates. Molecular evolution processes of sex chromosomes and sex determination provide a clue to tendency of sex-determining genes to appear on heterotypic sex chromosome.

[Molecular cloning and expression analysis of swamp eel (Monopterus albus) Hprt].

The enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) plays an important role in the purine salvage pathways. Full-length cDNA of swamp eel (Monopterus albus, the rice field eel) Hprt gene was obtained by the RACE method. The cDNA sequence was 1452 bp in length, which encoded a protein of 218 amino acids. Amino acid identities of Hprt between swamp eel and other vertebrates including human, mouse, chicken and zebrafish were more than 76.7%. Phylogenetic tree analysis based on the amino acid sequences showed that Hprt of swamp eel and zebrafish was in one clade. RT-PCR analysis showed ubiquitous expression pattern of swamp eel Hprt in adult tissues, suggesting that it has a constitutive function and is conserved during evolution.

[Analysis of regulatory regions of mammalian DMRT1 genes].

Dmrt1 gene is the first sex differentiation gene found conserved across animal phyla. It plays an important role in mammalian sexual differentiation. Sequence analysis of mammalian dmrt1 5' and 3' flanking regions showed that there were three and seven conserved regions (>60% homology), respectively. Dmrt1 promoter, 3' flanking region and coding sequence were amplified, cloned into expression vectors and transfected into both COS-7 and ST cells. Results indicated that both 5' and 3' flanking sequences could direct the expression of reporter gene gfp and dmrt1. However there was clearly a difference in expression efficiency between different cells. These results indicated that regulation of dmrt1 expression may be cell-specific and complex regulatory mechanism may be involved in dmrt1 expression.

Transcriptional diversity of DMRT1 (dsx- and mab3-related transcription factor 1) in human testis.

Recent advances in the evolutionary genetics of sex determination indicate that the only molecular similarity in sex determination found so far among phyla is between the fly doublesex, worm mab-3 and vertebrate DMRT1(dsx- and mab3-related transcription factor 1) /DMY genes. Each of these factors encodes a zinc-finger-like DNA-binding motif, DM domain. Insights into the evolution and functions of human DMRT1 gene could reveal evolutionary mechanisms of sexual development. Here we report the identification and characterization of multiple isoforms of human DMRT1 in the testis. These transcripts encode predicted proteins with 373, 275 and 175 amino acids and they were generated by alternative splicing at 3' region. Expression level of DMRT1a is higher than those of both DMRT1b and c, and the DMRT1c expression was the lowest in testis, based on comparisons of mean values from real-time fluorescent quantitative RT-PCR analysis. Both DMRT1b and c result from exonization of intronic sequences, including the exonization of an Alu element. A further search for Alu elements within the DMRT1 gene demonstrated that all 99 Alu elements are non-randomly distributed among the non-coding regions on both directions. These new characteristics of DMRT1 would have an important impact on the evolution of sexual development mechanisms.

[Inhibitory effect of human brain myelin basic protein on H2O2-induced apoptosis of human lung cancer cell line YTLMC-90].

BACKGROUND & OBJECTIVE: Human brain myelin basic protein (MBP) distributes in nervous system and other tissues extensively, and can be detected in many kinds of tumor cells, such as lung cancer, breast cancer, and neuroglioma. However, it has not been reported whether MBP is relevant to the activity of neural invasion of tumors and whether MBP plays a role in biological behaviors of human lung cancer cells. This study was to investigate the inhibitory effect of MBP on hydrogen peroxide (H2O2)-induced apoptosis of human lung cancer cell line YTLMC-90. METHODS: YTLMC-90 cells were transfected with plasmid pSVCEPMBPCAT containing MBP cDNA minigene (test group), or empty vector pSVCEPCAT, or received no transfection (control group), and exposed to H2O2. The expression of MBP in YTLMC-90 cells was detected by Western blot. Cell proliferation was measured by MTT assay. The morphologic and ultra-structural changes of apoptotic cells were observed by microscopy with fluorescent staining of acridine orange (AO) and electron microscopy. The DNA fragmentation was examined by agarose gel electrophoresis. RESULTS: After exposed to 200 micromol/L H2O2 for 24 h, the inhibitory rate of cell growth was significantly lower in test group than in empty vector group and control group (36.67% vs. 78.67% and 84.00%, P<0.001). The morphologic and biochemical changes of apoptotic cells, such as shrinkage of cytoplasm and nucleus, fragmentation of chromatin, and ladder pattern of DNA, were commonly observed in cells in control group, but these apoptotic features were not discovered in test group. CONCLUSION: MBP markedly inhibits H2O2 cytotoxicity to YTLMC-90 cells through promoting cell proliferation and antagonizing H2O2-induced apoptosis.

Molecular cloning of the rice field eel Nup93 with predominant expression in gonad and kidney.

Nucleoporins (Nups) are important components of nuclear pore complexes (NPCs). NPCs control gene expression, cells proliferation and differentiation by mediating exchange of cellular signal molecules on both nuclear and cytoplasmic sides. Using subtractive screening, 3'end fragment of Nup93 from the testis cDNA library of the rice field eel was obtained. Full-length cDNA of the gene was further cloned by degenerate PCR and 5'RACE methods. Sequence analysis indicated that the homology of the rice field eel Nup93 were 36.5% with yeast Nic96, 94.6% and 90.5% with Nup93 of zebrafish and human, respectively. Phylogenetic analysis showed that the rice field eel Nup93 fits with Nup93 of the other fishes. Real-time PCR result showed that expression of Nup93 in gonads and kidney were much higher than in other tissues, and different expression quantities among gonads of three sexes were also observed, suggesting that Nup93 may involve in gonad development.

[Characterization of the beta-actin gene of the rice field eel and its phylogeny in fish].

Beta-actin is a member of the actin family of genes,which play important roles in maintaining cytoskeletal structure, cell motility, cell division, intracellular movements and contractile processes. We report here the identification of a beta-actin cDNA of the rice field eel,a teleost fish with a characteristic of natural sex reversal. The cDNA sequence of this gene was 1860 bp in length,encoding a 375 amino acid protein. Amino acid identities of the beta-actin between the rice field eel and other vertebrates including human, chicken, and other fishes, were more than 98%. RT-PCR showed expression of the rice field eel beta-actin in testis, ovotestis, ovary, heart, liver, spleen and brain,suggesting a ubiquitous expression pattern. Phylogenetic tree including all beta-actin cds of teleost fishes was constructed,which suggested consistently that teleost beta-actin can be classified into four types,but no fish has been found contains all the four types of beta-actin gene in its genome. The results that suggest lineage-specific beta-actin loss might happen in the radical evolution of teleost fishes.

[Ribosomal protein genes highly expressed in swamp eel gonads].

8 cDNA clones have been isolated from a cDNA library prepared from swamp eel testies by macroarray. DNA sequence analysis and database search showed that they encode 8 proteins which are highly homologous to 40S ribosomal proteins S4,S9,S16,S17,S20 and 60S riobosomal proteins L7, L18a,L29. Phylogenetic trees (ML) based on ribosomal protein genes from swamp eel and other organisms has been reconstructed, which showed that ribosomal protein genes were highly conserved during evolution. These results suggested that ribosomal protein genes as house keeping genes may play roles in developmental regulation such as sexual differentiation and can also be used as markers for the study of molecular evolution.

[Phylogenetic tree and synteny of DMRT genes family of vertebrates].

Vertebrates contain a family of genes related to the Drosophila doublesex and C. elegans mab-3 genes, which encode transcription factors including a DNA-binding motif, DM domain. Evolution and function of different DMRT genes of vertebrates have not been understood yet,although some DM proteins are involved in sex determination, sexual differentiation and early embryonic development among different phyla. By genomic analysis of zebrafish and rat DMRT genes, all protein sequences of the vertebrate DMRTs were searched from gene databases and aligned. Phylogenetic tree of all these DMRT genes was reconstructed and evaluated by Bootstrap method. These DMRT genes were clustered into seven subfamilies. Results from analysis of gene structure and cluster organization of DMRT genes showed that synteny of DMRT genes of vertebrates were highly conserved among human, mouse, rat, fugu, medaka and zebrafish, with two syntenic groups, DMRT 1 approximately 3 and DMRT 5 approximately 6.

[Cloning of four members of giant panda Dmrt genes].

Sex determining genes Mab-3 of C. elegans and Doublesex of Drosophila contain a common DNA binding motif called DM (Doublesex and Mab-3) domain, both of which regulate similar aspects of sexual development. Human Doublesex-related gene DMRT1 has been identified, which also contains the conserved DM-related DNA-binding domain and plays an essential role in gonadal differentiation. We amplified genomic DNA of the giant panda using the DM degenerate primers and detected two bands, approximately 140 bp and 250 bp. After cloned into T-easy vector and sequenced, four sequences showed high homology with the DM domain. Amino acid sequence of the first clone is 100% identical with the Dmrt1 of human, mouse and pig, hence we named it as pDmrt1. The second clone is 96% identical with human DMRTB1, and the third one 100% with the Dmrt3 of mouse and medaka, which were named as pDmrtb1 and pDmrt3 respectively. The last sequence contains an intron of 116 bp within the DM domain, which encodes an amino acid sequence 100% identical with human DMRTC2, accordingly we named it as pDmrtc2. Based on similarities of amino acid sequences of the DM domain, Dmrt protein sequences from human, mouse and giant panda were included in a phylogenetic tree. They revealed seven distinct subgroups: Dmrt1, Dmrt2, Dmrt3, Dmrt4 (DMRTA1), Dmrt5 (DMRTA2), Dmrt6 (DMRTB1) and Dmrt7 (DMRTC2). Our results further reveal the unexpected complexity and the evolutionary conservation of the DM domain gene family in both invertebrates and vertebrates.

[Phylogenic analysis of the Sox gene family of vertebrate].

Sox genes of vertebrate are highly evolutionarily conserved, which encode different transcriptional factors involved in various developmental processes. Sox family is characterized by a sequence-specific DNA binding HMG-box containing about 79 amino acids. To realize the complexity of genes of the Sox family in the structures, functions and their evolutionary relationships, in the present study by utilizing all available complete nucleotide/protein sequence data of vertebrate Sox genes, we performed multi-sequence comparison and construction of phylogenic tree, and the grouping of Sox family members and the pattern of their molecular evolution was also analyzed.