Phosphorylation of PHO4 Protein by the PHO85-PAP1 Kinase Complex.

Homology comparison of the novel PAP1 protein indicated that PAP1 protein is highly homologous to PHO85-associated protein PHO80, PCL1 and PCL2. We constructed the fused HA-PAP1 gene in frame for immunoprecipitation with anti-HA monoclonal antibody. Coimmunoprecipitation of fused HA-PAP1 and PHO85 protein, which were translated in vitro, verified the association of PAP1 with PHO85 protein.

Cloning and Expression of a Novel PHO85 Associated Protein PAP1 Gene of Saccharomyces cerevisiae.

Yeast regulatory factor PHO85, which is a cyclin-dependent kinase (DCK), participates in the regulation of the cell cycle and the expression of the acid phosphatase gene. Using PHO85 as target, we have cloned from the yeast two-hybrid genomic library a novel gene (PAP1) with product which can asscociate with the PHO85 protein. The PAP1 gene including the 5apo; and 3apo; flanking sequence was cloned and sequenced.

A Novel cDNA Encoding Ubiquitin-conjugating Enzyme of Homo sapiens.

p53 interacts with a number of cellular proteins to form complexes which are probably crucial for its normal physiological function involving cell cycle control, gene regulation, cell differentiation, apoptosis and tumor suppression. To identify these proteins, we used the yeast two-hybrid system and screened a HeLa cDNA library. Six positive colonies were isolated from 1.

Molecular Mechanism of Yeast Phosphatase Gene Expression.

No Abstract

Functional Analysis of the Upstream Sequence of PHO81 Gene of Saccharomyces cerevisiae.

Deletion analysis on the fused PHO81-lacZ gene revealed two important regions in the upstream sequences of PHO81 gene -401 -289 bp and -1 012 -801 bp. They did not share higher similarity with the upstream regions of PHO5 and PHO84 gene, except that -401 -289 bp contains the 5'-CACGTG/T-3' motif, which was found among the upstream regions of PHO5 and PHO84 gene and was the core sequence of PHO4 binding site it also contains A/T-rich segments flanking the motif, which may be PHO2 binding sites. This suggests that the -401 -289 bp of the upstream region of PHO81 gene may contain upstream activation sequence (UAS), and -1 012 -801 bp may contain upstream enhancer sequence.

PHO4 and PHO2 Protein Interact with Upstream Sequence of PHO81 Gene.

PHO4 and PHO2 protein were overexpressed in E. coli and purified respectively. The gel retardation assays showed that PHO4 and PHO2 protein could bind to -401 -289 bp sequence of PHO81 gene promoter respectively.

Expression and Functional Analysis of Yeast PHO81 Ankyrin Repeats.

There are six ankyrin repeats in yeast transcriptional factor PHO81. The PHO81 ankyrin repeats fused with glutathione S-transferase (GST-ANK) was overexpressed in E. coli and it existed in inclusion body form.

Analysis of Interaction between PHO4 and PHO2 Protein by Real Time BIA.

Applying real time BIA(biomolecular interaction analysis) the interaction between yeast PHO4 and PHO2 protein was analyzed. Recombinant PHO4 protein was coupled at the sensor chip via amino group. 5 &mgr;mol/L of recombinant PHO2 and PHO2 mutants which were fused with glutathione S-transferase were injected respectively.

Analysis of phosphorylation of YJL084c, a yeast protein.

PCL6, PCL7(PAP1), and PHO80 belong to the PHO80 subfamily of PCLs (PHO85 cyclins), share high homology in protein sequences, and function with some similarity. YLR190w, the substrate of PCL7-PHO85, shares homology with YJL084c in a 140-amino-acid region. In addition, YJL084c was reported as a PCL6-binding protein.

[Phosphorylation of YLR190w by PAP1 PHO85 kinase complex].

A yeast two-hybrid screening by using PAP1 was performed to identify targets for PAP1-PHO85 cyclin-CDK complex. N-terminal fragment of protein YLR190w, a yeast gene encoding a 491 amino acids peptide, was identified, and its coding region was amplified by PCR. The interaction of PAP1 and YLR190w was confirmed by both two-hybrid assay and GST pull-down assay in vitro.

Analysis of Activation Activity of Yeast PHO2, PHO4 Protein and Their Interaction.

Both PHO2 and PHO4 are positive regulatory factors of yeast PHO5 gene. Here we show that the PHO2 fused to yeast transcriptional factor GAL4 DNA-binding domain activates the expression of the reporter gene (lacZ), and the lacZ activities were regulated by Pi concentration, therefore it could be suggested that there are acidic activation domains on the PHO2 protein. Acidic amino acid rich region of 287-326 aa in PHO2 is not a transcriptional activation domain.

Interaction of the Yeast PH02 Protein or Its Mutants with the PHO5 UAS in vitro.

The GST gene fusion system was used to express PHO2 gene and its mutants in E. coli Gel retardation assays showed that PHO2 fusion protein can bind to the upstream activation sequence (UAS) of the acid phosphatase gene PHO5. The homeodomain of PHO2 protein has such structure as alpha-helix 2-beta-turn-alpha-helix 3, which acts as the DNA binding domain of the transcriptional factor.

Studies on the Potential Phosphorylation Sites of the Yeast PHO2 Factor.

We report here that PHO2 protein is also phosphorylated by an unidentified protein kinase. A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cc2/CDC28-related kinase in the PHO2 protein led to the complete loss of its ability to activate the transcription of PHO5 gene. Further work showed that Pro-231 to Ser mutation inactivated PHO2 protein as well, while Ser-230 to Asp mutation did not affect PHO2 activity.

The Effect of Yeast Transcriptional Factor PHO2 on the Gene Expression of PHO5, HIS4 and HO.

Yeast PHO2 protein plays a role in the expression of several different genes and acts as a multiple global activator. Here we report the comparison of the effect of PHO2 protein on the expression of PHO5, HIS4 and Ho genes. In the PHO2 defective yeast strain, PHO5 gene could not be depressed in low Pi and the expression of the HIS4 and HO genes was decrease to 25% and 40% of the normal level respectively.

Functional Domains of the Regulatory Factor PHO81 of Saccharomyces cerevisiae.

It is found that minor changes around the basic motif (88-160) and the acidic motif (771-810) of the PHO81 protein can lead to the constitutive expression of the acid phosphatase gene (PHO5), and the two motifs work cooperatively. The PHO81 protein has six ankyrin repeats, which are the recognition sites of PHO81 protein with the PHO80-PHO85 protein complex. It is found that the ankyrin repeats 1,2,4,5 and 6 are important for the PHO81 protein, but the deletion of Pro(509) and Leu(510) in ankyrin repeat 3 does not affect the PHO81 protein.

A Structure-function Analysis of the Human Ciliary Neurotrophic Factor.

The ciliary neurotrophic factor (CNTF) plays a very important role in the development and regeneration of the nervous system. In this study, the prediction of secondary structure and the hydrophobicity analysis of human CNTF were performed according to the amino acid sequence deduced from the nucleotide sequence of the cDNA. Based on the results of the prediction of structure, the human CNTF gene was modified by insertion and deletion mutagenesis.