The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant ...The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant sequence identity to the HMG-box family of proteins. A multiple sequence alignment of several α-domains and various structurally determined HMG-box domains has revealed that both domains possess very similar structural and functional residues. We found that the basic amino acids of the N-terminal loop, the intercalating hydrophobic residues of the first helix, and the hydrophobic residues required for interactions within the core of the protein are remarkably conserved in α-domains and HMG-box proteins. Our generated molecular models suggest that the first and third helix will be shorter and that the HMG-box core is not an isolated domain. The region beyond the conserved HMG-box motif contains an extended helical region for about 20 - 30 amino acids. Structural models generated by comparative modeling and ab initio modeling reveal that this region will add two or more additional α-helices and will make significant contacts to helix III, II and I of the HMG-box core. We were able to illustrate how the extended α-domain would bind to DNA by merging of the α-domain and the LEF-1/DNA complex. The models we are reporting will be helpful in understanding how MATα1 binds to DNA with its partner MCM1 and activates transcription of α-specific genes. These models will also aid in future biophysical studies of MATα1 including the crystallization and structure determination.展开更多
The objective of these studies were to identify ruminal yeast in varying ratios of roughage to concentrate in TMR diets in order to explore yeast diversity by using molecular technique with similarity of rDNA sequence...The objective of these studies were to identify ruminal yeast in varying ratios of roughage to concentrate in TMR diets in order to explore yeast diversity by using molecular technique with similarity of rDNA sequence. The experiment was assigned to four 98.6% of cross bred Holstein Friesian heifers with 2 levels and two replicates of roughage to concentrate ratios as： 10：90 （T1） and 50：50 （T2）. The experimental period was 14 days. Rumen fluid sample was collected by stomach tube for total DNA extraction by using silica gel method, and analysis of quantity and quality of DNA by Nanovue and agarose gel electrophoresis. The divergent DI/D2 domain of 26S rDNA was amplified by primers NL-1（5＇-GCA TAT CAA TAA GCG GAG GAA AAG-Y） and NL4 （5＇-GGT CCG TGT TTCAAG ACG G-3＇） by polymerase chain reaction （PCR）. The nucleotide sequences of D l/D2 domain of 26S rDNA were determined using PCR products. Generated sequences were aligned with related species by using the CLUSTAL W. The result showed that an average dry matter intake of TI was 7.00 kg/d and T2 was 6.99 kg/d. DNA concentrate from TIRI, TIR2, T2RI and T2R2 were 106, 131.5, 84 and 182.5 ng//aL, respectively. The purity of DNA was 1.57, 1.76, 1.78 and 1.86, respectively. The divergent D1/D2 domain of 26S rDNA of treatment could be amplified for T1R1 and T2R1 but could not for T1R2 and T2R2. The sequences of D1/D2 domain of 26S rDNA were compared with nucleotide database by BLAST programs （http：//www.ncbi.nlrn.nih.gov/BLAST）, the T2RI yeast-strain was closest to Yarrowia lipolytica. However, yeast strain in T1R1 could not be specifically identified because D 1/132 domain of 26S rDNA seem to represent variable region with number of nucleotide sequence showing 2-3 substitution from known species. The phylogenetic tree based on the sequences of the DI/D2 domain of 26S rDNA showed that TIR! was related to Pichia and Candida （96%） and T2R1 was related to Yarrowia lypolytica （100%）. This study indicated that ruminal yeast strains could be found varying in different ratio of roughage to concentrate.展开更多
Metallothioneins (MTs) are a group of low molecular mass and cysteine-rich proteins that can chelate heavy-metal ions. In this paper,Northern blot analysis was used to investigate the influence of lead stress on the e...Metallothioneins (MTs) are a group of low molecular mass and cysteine-rich proteins that can chelate heavy-metal ions. In this paper,Northern blot analysis was used to investigate the influence of lead stress on the expression patterns of 10 rice class I MT genes (OsMT-Is) in rice seedlings. With the ex-ception of OsMT-I-3b,the data demonstrate dynamic changes of 9 OsMT-I transcripts in response to Pb2+ treatment in rice seedling roots. Of these genes,transcription of OsMT-I-1a,OsMT-I-1b,OsMT-I-2c,OsMT-I-4a,OsMT-I-4b and OsMT-I-4c increased significantly,while transcription of OsMT-I-2a and OsMT-I-3a increased marginally. In contrast,the expression of OsMT-I-2b was inhibited. Pb2+ induced the expression of 6 OsMT-I genes in seedling shoots,but had no obvious effects on the expression of OsMT-I-1a,OsMT-I-1b,OsMT-I-4a and OsMT-I-4b. All the 10 OsMT-Is had enhanced lead tolerance when heterologously expressed in lead-sensitive yeast mutant cells. These results provide an expression profile of the rice MT gene family in response to Pb2+ stress in rice seedlings and demonstrate in-creased lead tolerance in sensitive yeast mutant cells expressing OsMT-Is. This study lays a foundation for further analysis of the role of the rice MT gene family in respond to Pb2+ stress.展开更多
Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells...Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.展开更多
文摘The yeast MATα1 is required for the activation of α-specific genes in Saccharomyces cerevisiae and thus confers the α-cell identity of the yeast. MATα1 contains a domain called the α-domain which has significant sequence identity to the HMG-box family of proteins. A multiple sequence alignment of several α-domains and various structurally determined HMG-box domains has revealed that both domains possess very similar structural and functional residues. We found that the basic amino acids of the N-terminal loop, the intercalating hydrophobic residues of the first helix, and the hydrophobic residues required for interactions within the core of the protein are remarkably conserved in α-domains and HMG-box proteins. Our generated molecular models suggest that the first and third helix will be shorter and that the HMG-box core is not an isolated domain. The region beyond the conserved HMG-box motif contains an extended helical region for about 20 - 30 amino acids. Structural models generated by comparative modeling and ab initio modeling reveal that this region will add two or more additional α-helices and will make significant contacts to helix III, II and I of the HMG-box core. We were able to illustrate how the extended α-domain would bind to DNA by merging of the α-domain and the LEF-1/DNA complex. The models we are reporting will be helpful in understanding how MATα1 binds to DNA with its partner MCM1 and activates transcription of α-specific genes. These models will also aid in future biophysical studies of MATα1 including the crystallization and structure determination.
文摘The objective of these studies were to identify ruminal yeast in varying ratios of roughage to concentrate in TMR diets in order to explore yeast diversity by using molecular technique with similarity of rDNA sequence. The experiment was assigned to four 98.6% of cross bred Holstein Friesian heifers with 2 levels and two replicates of roughage to concentrate ratios as： 10：90 （T1） and 50：50 （T2）. The experimental period was 14 days. Rumen fluid sample was collected by stomach tube for total DNA extraction by using silica gel method, and analysis of quantity and quality of DNA by Nanovue and agarose gel electrophoresis. The divergent DI/D2 domain of 26S rDNA was amplified by primers NL-1（5＇-GCA TAT CAA TAA GCG GAG GAA AAG-Y） and NL4 （5＇-GGT CCG TGT TTCAAG ACG G-3＇） by polymerase chain reaction （PCR）. The nucleotide sequences of D l/D2 domain of 26S rDNA were determined using PCR products. Generated sequences were aligned with related species by using the CLUSTAL W. The result showed that an average dry matter intake of TI was 7.00 kg/d and T2 was 6.99 kg/d. DNA concentrate from TIRI, TIR2, T2RI and T2R2 were 106, 131.5, 84 and 182.5 ng//aL, respectively. The purity of DNA was 1.57, 1.76, 1.78 and 1.86, respectively. The divergent D1/D2 domain of 26S rDNA of treatment could be amplified for T1R1 and T2R1 but could not for T1R2 and T2R2. The sequences of D1/D2 domain of 26S rDNA were compared with nucleotide database by BLAST programs （http：//www.ncbi.nlrn.nih.gov/BLAST）, the T2RI yeast-strain was closest to Yarrowia lipolytica. However, yeast strain in T1R1 could not be specifically identified because D 1/132 domain of 26S rDNA seem to represent variable region with number of nucleotide sequence showing 2-3 substitution from known species. The phylogenetic tree based on the sequences of the DI/D2 domain of 26S rDNA showed that TIR! was related to Pichia and Candida （96%） and T2R1 was related to Yarrowia lypolytica （100%）. This study indicated that ruminal yeast strains could be found varying in different ratio of roughage to concentrate.
基金Supported by the State Key Basic Research and Development Plan of China (Grant No. 2006CB101706)the National Natural Science Foundation of China (Grant Nos. 30270753 & 30370804)
文摘Metallothioneins (MTs) are a group of low molecular mass and cysteine-rich proteins that can chelate heavy-metal ions. In this paper,Northern blot analysis was used to investigate the influence of lead stress on the expression patterns of 10 rice class I MT genes (OsMT-Is) in rice seedlings. With the ex-ception of OsMT-I-3b,the data demonstrate dynamic changes of 9 OsMT-I transcripts in response to Pb2+ treatment in rice seedling roots. Of these genes,transcription of OsMT-I-1a,OsMT-I-1b,OsMT-I-2c,OsMT-I-4a,OsMT-I-4b and OsMT-I-4c increased significantly,while transcription of OsMT-I-2a and OsMT-I-3a increased marginally. In contrast,the expression of OsMT-I-2b was inhibited. Pb2+ induced the expression of 6 OsMT-I genes in seedling shoots,but had no obvious effects on the expression of OsMT-I-1a,OsMT-I-1b,OsMT-I-4a and OsMT-I-4b. All the 10 OsMT-Is had enhanced lead tolerance when heterologously expressed in lead-sensitive yeast mutant cells. These results provide an expression profile of the rice MT gene family in response to Pb2+ stress in rice seedlings and demonstrate in-creased lead tolerance in sensitive yeast mutant cells expressing OsMT-Is. This study lays a foundation for further analysis of the role of the rice MT gene family in respond to Pb2+ stress.
文摘Yeast high stable plasmid vector pHC11 was constructed by introducing pEMBL Yi27 cleaved with SmaI into the SnaBI site of intact 2 μm plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 μm plasmid. The human interferon αA (IFN αA) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. Tbe data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN αA biological activity was 2.6×10^(10) units per liter, demonstrating that high-level expression and secretion of IFN αA were achieved in yeast by using the stable vector pHC11.
