Biomedical subjects
Publications and source records attributed to H Ronne.
In many organisms, glucose represses genes that are used to metabolize other carbon sources. Work in yeast and filamentous fungi has revealed a mechanism for glucose repression in eukaryotes that is different from that found in bacteria. Zinc finger proteins, such as Mig1 and CREA, that bind GC-boxes play a key role in mediating this response.
BTF3 is a human protein that is thought to be involved in transcription by RNA polymerase II [Zheng et al., Cell 50, 361-368, 1987]. A yeast homologue of BTF3, Egd1p, has been identified by its ability to enhance DNA binding of the Gal4p activator [Parthun et al., Mol. Cell. Biol. 12, 5683-5689, 1992]. We have cloned a second yeast gene, BTT1, which also encodes a BTF3 homologue. Btt1p and Egd1p are highly similar in sequence, which suggests that they are duplicated proteins with similar functions. Gene disruptions were used to investigate the function of the two proteins. Consistent with published results, we found that loss of EGD1 causes a minor defect in GAL gene induction. Loss of BTT1 has little if any effect. Surprisingly, we found that cells which lack both genes instead express the GAL1 and GAL10 mRNAs at much higher levels than wild type cells. This suggests that BTF3 really plays a negative role in GAL gene expression. Further experiments revealed that expression of the ACT1 and SSO1 genes also is elevated in cells that lack EGD1 and BTT1. In contrast, expression of rRNA and tRNA was not affected. We conclude that Btt1p and Egd1p have redundant functions in vivo, and that they exert a negative effect on the expression of several genes that are transcribed by RNA polymerase II.
Protein phosphatase 2A (PP2A) is an essential enzyme which is present in all eukaryotic cells. PP2A has been implicated in regulating various metabolic processes and also in the control of cell cycle progression. In the yeast Saccharomyces cerevisiae, the catalytic subunit of PP2A is encoded by two duplicated genes, PPH21 and PPH22. A third related gene, PPH3, also contributes some PP2A activity. We have used a yeast strain in which a single functional PP2A gene is expressed from a regulated promoter to screen for high copy number suppressors of PP2A depletion. A new gene was cloned, PAM1 (PP2A multicopy suppressor), which in high copy number can bypass the need for a PP2A catalytic subunit. The PAM1 gene encodes a hydrophilic 93-kDa protein that contains two coiled coil motifs and has a highly basic C-terminal tail. High level overexpression of PAM1 inhibits growth and induces a filamentous phenotype.
MIG1 is a zinc finger protein that mediates glucose repression in the yeast Saccharomyces cerevisiae. MIG1 is related to the mammalian Krox/Egr, Wilms' tumor, and Sp1 finger proteins. It has two fingers and binds to a GCGGGG motif that resembles the GC boxes recognized by these mammalian proteins. We have performed a complete saturation mutagenesis of a natural MIG1 site in order to elucidate its binding specificity. We found that only three mutations within the GC box retain the ability to bind MIG1: G1 to C, C2 to T, and G5 to A. This result is consistent with current models for zinc finger-DNA binding, which assume that the sequence specificity is determined by base triplet recognition within the GC box. Surprisingly, we found that an AT-rich region 5' to the GC box also is important for MIG1 binding. This AT box is present in all natural MIG1 sites, and it is protected by MIG1 in DNase I footprints. However, the AT box differs from the GC box in that no single base within it is essential for binding. Instead, the AT-rich nature of this sequence seems to be crucial. The fact that AT-rich sequences are known to increase DNA flexibility prompted us to test whether MIG1 bends DNA. We found that binding of MIG1 is associated with bending within the AT box. We conclude that DNA binding by a simple zinc finger protein such as MIG1 can involve both recognition of the GC box and flanking sequence preferences that may reflect local DNA bendability.
A gene encoding transketolase, TKL1, was cloned from the budding yeast Saccharomyces cerevisiae using oligonucleotide primers derived from protein sequence data. The TKL1 sequence predicts a 74-kDa polypeptide which is related to other transketolases. A sequence comparison revealed that the transketolases can be subdivided into three evolutionary branches. We also found that the transketolases are related to another vitamin B1-dependent enzyme: the E1 subunit of pyruvate dehydrogenase from Escherichia coli. Gene disruption and overexpression experiments were used to investigate the function of transketolase in yeast. We found that growth on fermentable carbon sources, but not on gluconeogenic carbon sources, is reduced in cells disrupted for TKL1. This suggests that the glycolytic efficiency is impaired. Growth on fermentable carbon sources is also reduced in cells that overexpress TKL1. Finally, we found that cells disrupted for TKL1 are unable to grow in the absence of aromatic amino acids. This is most likely due to the fact that transketolase is required for the synthesis of erythrose-4-P, a precursor of the aromatic amino acids.
The yeast SEC1 gene encodes a hydrophilic protein that functions at the terminal stage in secretion. We have cloned two yeast genes, SSO1 and SSO2, which in high copy number can suppress sec1 mutations and also mutations in several other late acting SEC genes, such as SEC3, SEC5, SEC9 and SEC15. SSO1 and SSO2 encode small proteins with N-terminal hydrophilic domains and C-terminal hydrophobic tails. The two proteins are 72% identical in sequence and together perform an essential function late in secretion. Sso1p and Sso2p show significant sequence similarity to six other proteins. Two of these, Sed5p and Pep12p, are yeast proteins that function in transport from ER to Golgi and from Golgi to the vacuole, respectively. Also related to Sso1p and Sso2p are three mammalian proteins: epimorphin, syntaxin A/HPC-1 and syntaxin B. A nematode cDNA product also belongs to the new protein family. The new protein family is thus present in a wide variety of eukaryotic cells, where its members function at different stages in vesicular transport.
We have cloned a yeast gene, SKO1, which in high copy number suppresses lethal overexpression of cAMP-dependent protein kinase. SKO1 encodes a bZIP protein that binds to the CRE motif, TGACGTCA. We found that SKO1 also binds to a CRE-like site in SUC2, a yeast gene encoding invertase which is under positive control by cAMP. A disruption of the SKO1 gene causes a partial derepression of SUC2, indicating that SKO1 is a negative regulator of the SUC2 gene. SKO1 interacts positively with MIG1, a zinc finger protein that mediates glucose repression of SUC2. A kinetic analysis revealed a complex regulation of the SUC2 mRNA in response to glucose. First, MIG1 mediates a rapid and strong repression of SUC2, which is complete within 10 minutes. Second, a MIG1-independent process causes a further slow reduction in the mRNA. Third, in the absence of MIG1, there is also a rapid but transient glucose induction of the SUC2 mRNA. This induction is correlated with a transient loss of SKO1-dependent repression.
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Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.
We have cloned a gene encoding a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae by low stringency hybridization to PPA1, the yeast gene for cytoplasmic PPase. The new gene, PPA2, is located on chromosome 13 and encodes a protein whose sequence is 49% identical to the cytoplasmic enzyme. The protein differs from cytoplasmic PPase in that it has a leader sequence enriched in basic and hydroxylated residues, which is typically found in mitochondrial proteins. Yeast cells overproducing PPA2 had a 47-fold increase in mitochondrial PPase activity. This activity was further stimulated 3-fold by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which suggests that PPA2 is part of an energy-linked enzyme. Using gene disruptions, we found that PPA1 is required for cell growth. In contrast, cells disrupted for PPA2 are viable, but unable to grow on respiratory carbon sources. Fluorescence microscopy revealed that these cells have lost their mitochondrial DNA. We conclude that the mitochondrial PPase encoded by PPA2 is essential for mitochondrial function and maintenance of the mitochondrial genome.
Glucose repression is a global regulatory mechanism in yeast. We have investigated how glucose regulates the GAL genes, which are required for galactose fermentation. We found that the GAL genes are controlled by a transcriptional cascade. Thus, GAL4, which encodes an activator of the GAL genes, is repressed by MIG1, a zinc finger protein that binds to the GAL4 promoter. MIG1 has a dual role in control of the GAL genes, since MIG1 also binds to the promoter of GAL1, a gene regulated by GAL4. A disruption of MIG1 interacts synergistically with a disruption of GAL80, a gene involved in galactose induction. This suggests that the MIG1-dependent response to glucose is amplified by down-regulation of the induction pathway.
We have cloned three genes for protein phosphatases in the yeast Saccharomyces cerevisiae. Two of the genes, PPH21 and PPH22, encode highly similar proteins that are homologs of the mammalian protein phosphatase 2A (PP2A), while the third gene, PPH3, encodes a new PP2A-related protein. Disruptions of either PPH21 or PPH22 had no effects, but spores disrupted for both genes produced very small colonies with few surviving cells. We conclude that PP2A performs an important function in yeast cells. A disruption of the third gene, PPH3, did not in itself affect growth, but it completely prevented growth of spores disrupted for both PPH21 and PPH22. Thus, PPH3 provides some PP2A-complementing activity which allows for a limited growth of PP2A-deficient cells. Strains were constructed in which we could study the phenotypes caused by either excess PP2A or total PP2A depletion. We found that the level of PP2A activity has dramatic effects on cell shape. PP2A-depleted cells develop an abnormal pear-shaped morphology which is particularly pronounced in the growing bud. In contrast, overexpression of PP2A produces more elongated cells, and high-level overexpression causes a balloonlike phenotype with huge swollen cells filled by large vacuoles.
We have cloned a yeast gene, MIG1, which encodes a C2H2 zinc finger protein involved in glucose repression. The fingers of MIG1 are very similar to those present in the mammalian Egr finger proteins, which are induced during the early growth response, and also to the finger protein encoded by a human gene that is deleted in Wilms' tumour cells. MIG1 protein binds to two sites in the upstream region of SUC2, a yeast gene that is repressed by glucose. The MIG1 sites closely resemble the sequence recognized by the Egr proteins. Thus, finger proteins that are similar in both amino acid sequence and DNA specificity are involved in the response of yeast to glucose, and in the mammalian early growth response.
We have cloned and sequenced the GAL2 gene of Saccharomyces cerevisiae, which encodes galactose permease. The GAL2 protein is related to the yeast glucose transporter encoded by the SNF3 gene, and also to mammalian and bacterial sugar permeases. Like the other members of this protein family, GAL2 has twelve hydrophobic segments that are separated by loops of charged amino acids. A comparison of different members of this protein family shows that those parts of the polypeptides thought to be on the cytoplasmic side of the cell membrane, are more conserved than other parts of the molecules.
The major histocompatibility complex of the mouse contains numerous class I genes, most of which are encoded in the Qa and Tla regions. By hybridizations, the murine class I genes have been classified into three major families (Rogers, J. H. (1985a) Immunogenetics 21, 343-353). As yet, complete sequences are available only for members of family 1 (several H-2 and Qa genes) or family 2 (the pseudoallelic Tla genes T3b and T13c). We here present the complete nucleotide sequence of a gene from the Tla region that belongs to family 3. This gene, T2Aa, is a pseudogene by several criteria. The general structure of the gene is nonetheless well preserved. A comparison of the T2Aa sequence to those of other murine class I genes confirms the classification into three gene families. Members of gene families 2 and 3, located in the Tla region, are no more similar to each other than to family 1 (the H-2 and Qa2,3 genes). This suggests that families 2 and 3 were both created by ancient duplications of the functionally important family 1 genes. The fact that families 2 and 3 have diverged extensively both from family 1 and from each other may suggest that they are devoid of function.
In yeast meiosis, ascosporal colonies are sometimes sectored for a marker--i.e., half the colony has one allele and half has the other. This is interpreted as replicative resolution of heteroduplex DNA (hDNA) formed as a recombination intermediate. We have looked for similar evidence of hDNA formation during mitotic recombination between two repeated sequences on the same chromosome. The two repeats, an ochre suppressor and a wild-type tRNA gene, are separated by plasmid DNA and the URA3 marker. Recombination between the repeats excises the URA3 gene and one copy of the repeat, leaving either the wild-type tRNA or the suppressor on the chromosome. A red/white color assay is used to distinguish between the two. We find that some colonies that have lost the URA3 gene are sectored for the suppressor. This suggests that hDNA is formed across the anticodon during the recombination event and then resolved by replication. The disruption of either of two genes involved in recombination and repair, RAD1 and RAD52, does not significantly alter the frequency of sectored colony formation during plasmid excision.
The complete amino acid sequence of a cellular retinol-binding protein (CRBP) has been determined for the first time. The primary structure of rat liver CRBP was elucidated by analyses of cyanogen bromide fragments and peptides obtained by tryptic and thermolytic digestions. The single polypeptide chain of rat CRBP consists of 134 amino acid residues. Under reducing conditions, CRBP exists as a monomer, but, in the absence of reducing agents, dimers and multimers of the protein emerge. This is explained by the observation that CRBP contains 3 cysteines, one of which seems to be highly reactive. Whether CRBP contains a disulfide bond is not yet established. The present data extend the previously described homology between CRBP and a family of low molecular weight proteins, all members of which may bind hydrophobic ligands. Since some of these proteins apparently display intracellular transport functions, a similar role for CRBP is envisaged.
We have isolated a class I gene from the TL region of the A/J mouse. The gene, T2A, is a homologue of the C57BL/10 mouse gene T2. In the process of mapping this gene we screened a number of BALB/c class I cosmid clusters with a T2A flanking probe. Several of the hybridizing clusters were found to contain identical DNA segments and could therefore be linked together into one single BALB/c TL region which appears to be identical to the TL region of the C57BL/10 mouse. However, two of the hybridizing clusters do not overlap with the C57BL/10 TL region. It appears that these two clusters represent a partial duplication of the TL region in the BALB/c mouse.