Advance in research on spinocerebellar ataxia 17 / 中华医学遗传学杂志

Hereditary spinocerebellar ataxia type 17 (SCA17) is an autosomal dominantly inherited progressive degenerative disease of the nervous system. Also known as Huntington's disease-like 4(HDL4), SCA17 mainly features ataxia, muscle dystonia and psychiatric symptoms. The gene predisposing to SCA17 has been mapped and cloned, which encodes a TATA-binding protein (TBP). A CAG repeat expansion in the coding region of TBP gene can cause polyglutamine chain extension in the protein. This paper reviews recent progress in the research on SCA17 in regard to its clinical, etiology, pathology and pathogenesis.

Age estimation using content of sjTREC in human peripheral blood / 法医学杂志

OBJECTIVE@#To determine and verify the correlation formula of age estimation using the content of signal joint T-cell receptor excision DNA circle (sjTREC) in human peripheral blood and to discuss its application value in forensic biological practice.@*METHODS@#The samples of peripheral blood stains were collected from 30 healthy unrelated individuals whose ages were known. The DNAs were extracted from the samples stored at room temperature after 4 weeks. The content of sjTREC was measured by real-time fluorescent quantitative PCR technique, and the TATA box binding protein (TBP) was selected as reference genes. The age of each sample was predicted with the formula which was Age = -7.181 5 Y-42.458 +/- 9.42 (Y = dCtTBP-sjTREC), and the result was compared with the real age of each individual to determine the accuracy of the formula.@*RESULTS@#sjTREC and TBP gene were detectable in all 30 samples of peripheral blood. The contents of sjTREC in human peripheral blood showed a decreasing tendency with aging. The accuracy rate for the age estimation by this method was 76.67%.@*CONCLUSION@#The method for the age estimation with the content of sjTREC was simple, fast, sensitive, and good species specific with important potential application prospect.

The effects of TATA-box in CYC1 promoter on the reporter gene regulated by ERE in the recombinant yeast cell / 生物医学工程学杂志

The complementary oligonucleotides, each with two consensus estrogen response element (ERE)-sequences and 5'-Hind III and 3'-Sph I sticky ends were artificially synthesized. A solution with both the complementary DNA sequences was heated to 95'C and cooled down to room temperature to form double strand DNA (dsDNA). The set was cloned into the corresponding sites of CYC1 promoter of the pERE-CYC-yEGFP to yield pERE-CYCalpha-yEGFP vector. The two different reporter vectors, pERE-CYC-yEGFP and pERE-CYCalpha-yEGFP, the 2ERE, were placed in the CYC1 promoter. The former promoter downstream ERE contains alpha and beta-TATA boxes and the latter has only alpha-TATA box. The two different reporter vectors were transformed into the yeast cells that express human estrogen receptor alpha (ERalpha). Incubation of the recombinant yeasts with the six estrogenic compounds for 4 hours showed that the recombinant cell containing pERE-CYCalpha-yEGFP would give very poor dose-response curves, in contrast to the recombinant cell containing pERE-CYC-yEGFP which produced well-shaped dose-response curves. So it is necessary for this bioassay that alpha and beta-TATA boxes in the minimal CYC1 promoter when the promoter is used as a rapid and high throughput system for screening estrogenic chemical products.

Effects of mutational sptl5 gene to xylose utilization of Saccharomyces cerevisiae / 生物工程学报

We used genetic methods to get a mutational spt15 gene from the recombinant strain Saccharomyces cerevisiae YPH499-3, screened by global transcription machinery engineering (gTME) approach. We transformed the gene into the original strain Saccharomyces cerevisiae YPH499 using the vector pYX212, then got a new recombinant strain. We studied the characteristic of this strain and found that it could metabolize xylose and co-ferment xylose and glucose. Under the fermentation condition of 30 degrees C, 200 r/min, 72 h, the utilization ratio of xylose was 82.0%, with 32.4% of ethanol yield when the carbon source in the media was 50 g/L xylose, while the utilization ratio of xylose and glucose was 80.4% and 100% respectively, with the 31.4% of ethanol yield when the carbon source was 50 g/L glucose/xylose (1:1). Meanwhile, the concentration of the by-product xylitol was very low. This study demonstrates the effect which the forward mutation of spt15 gene makes to the co-fermentation of xylose and glucose to ethanol by Saccharomyces cerevisiae.

Regulation of activity of the yeast TATA-binding protein through intra-molecular interactions.

Dimerization is proposed to be a regulatory mechanism for TATA-binding protein (TBP) activity both in vitro and in vivo. The reversible dimer-monomer transition of TBP is influenced by the buffer conditions in vitro. Using in vitro chemical cross-linking, we found yeast TBP (yTBP) to be largely monomeric in the presence of the divalent cation Mg2+, even at high salt concentrations. Apparent molecular mass of yTBP at high salt with Mg2+, run through a gel filtration column, was close to that of monomeric yTBP. Lowering the monovalent ionic concentration in the absence of Mg2+, resulted in dimerization of TBP. Effect of Mg2+ was seen at two different levels: at higher TBP concentrations, it suppressed the TBP dimerization and at lower TBP levels, it helped keep TBP monomers in active conformation (competent for binding TATA box), resulting in enhanced TBP-TATA complex formation in the presence of increasing Mg2+. At both the levels, activity of the full-length TBP in the presence of Mg2+ was like that reported for the truncated C-terminal domain of TBP from which the N-terminus is removed. Therefore for full-length TBP, intra-molecular interactions can regulate its activity via a similar mechanism.

The transcriptional activator GAL4-VP16 regulates the intra-molecular interactions of the TATA-binding protein.

Binding characteristics of yeast TATA-binding protein (yTBP) over five oligomers having different TATA variants and lacking a UASGAL, showed that TATA-binding protein (TBP)-TATA complex gets stabilized in the presence of the acidic activator GAL4-VP16. Activator also greatly suppressed the non-specific TBP-DNA complex formation. The effects were more pronounced over weaker TATA boxes. Activator also reduced the TBP dimer levels both in vitro and in vivo, suggesting the dimer may be a direct target of transcriptional activators. The transcriptional activator facilitated the dimer to monomer transition and activated monomers further to help TBP bind even the weaker TATA boxes stably. The overall stimulatory effect of the GAL4-VP16 on the TBP-TATA complex formation resembles the known effects of removal of the N-terminus of TBP on its activity, suggesting that the activator directly targets the N-terminus of TBP and facilitates its binding to the TATA box.

Cis-acting elements, CArG-, E-, CCAAT- and TATA-boxes may be involved in sexually regulated gene transcription in Schistosoma mansoni

Schistosomes undergo various morphological and metabolic changes during their development, reflected in a finely tuned regulation of protein and/or gene expression. The mechanisms involved in the control of gene expression during the development of the parasite are not understood. Two actin genes had been previously cloned and observed to be differentially expressed during the maturation of the parasite. The SmAct gene contains four putative cis-regulatory elements (TATA-, CCAAT-, E- and CArG-boxes). Our objective was to investigate in greater detail the expression pattern of two actin genes and verify if the binding of nuclear proteins to the promoter elements of SmAct correlated with the expression profile observed. We detected little variation in the expression of actin genes during the first seven days of schistosomula culture in vitro. However, we observed significantly higher levels of expression in males compared to female adults. CArG and CCAAT elements bound to a greater extent and formed distinct complexes with male in comparison to female nuclear extracts. In contrast, female extracts bound weakly to the E-box probe while no binding was observed with male extracts. Taken together these results describe cis-acting elements that appear to be involved in sexually regulated gene expression in Schistosoma mansoni

A Human Immunodeficiency Virus Type 1 (HIV-1) Tat Cofactor Absent in Rodent Cells is a TAR-associated Factor

BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.

A Human Immunodeficiency Virus Type 1 (HIV-1) Tat Cofactor Absent in Rodent Cells is a TAR-associated Factor

BACKGROUND: Although Tat plays a role as a potent transactivator in the viral gene expression from the Human Immunodeficiency Virus type 1 long terminal repeat (HIV-1 LTR), it does not function efficiently in rodent cells implying the absence of a human specific factor essential for Tat-medicated transactivation in rodent cells. In previous experiments, we demonstrated that one of chimeric forms of TAR (transacting responsive element) of HIV-1 LTR compensated the restriction in rodent cells. METHODS: To characterize the nature of the compensation, we tested the effects of several upstream binding factors of HIV-1 LTR by simple substitution, and also examined the role of the configuration of the upstream binding factor(s) indirectly by constructing spacing mutants that contained insertions between Sp1 and TATA box on Tat-mediated transactivation. RESULTS: Human Sp1 had no effect whereas its associated factors displayed differential effects in human and rodent cells. In addition, none of the spacing mutants tested overcame the restriction in rodent cells. Rather, when the secondary structure of the chimeric HIV-1 TAR construct was destroyed, the compensation in rodent cells was disappeared. Interestingly, the proper interaction between Sp1 and TATA box binding proteins, which is essential for Tat-dependent transcription, was dispensable in rodent cells. CONCLUSION: This result suggests that the human-specific Tat cofactor acts to allow Tat to interact effectively in a ribonucleoprotein complex that includes Tat, cellular factors, and TAR RNA, rather than be associated with the HIV-1 LTR upstream DNA binding factors.