Biomedical subjects

C M Egli

Publications and source records attributed to C M Egli.

11 recordsLinked to original sources

PubMed1999

A single point mutation in the yeast TRP4 gene affects efficiency of mRNA 3' end processing and alters selection of the poly(A) site.

The yeast TRP4 mRNA 3' end formation element is a bidirectional element which functions in both orien-tations in an artificial in vivo test system. In this study, the role of 3' end formation was analysed in the context of the entire TRP4 gene. The 3' untranslated region (3'UTR) of TRP4 was altered and changes were analysed for their influence on TRP4 gene expression. The 3'UTR in reverse orientation was fully functional and did not affect TRP4 gene expression. Exchanging the TRP4 3'UTR by the bidirectional ARO4 or the unidirectional GCN4 3' end formation element allowed efficient gene expression. Deletion of the entire TRP4 3'UTR resulted in 70% reduction of TRP4 mRNA and 50% reduced specific Trp4 enzyme activity in comparison to wild-type. A single point mutation within the TRP4 3'UTR caused the same effect on gene expression. This point mutation did not only affect the efficiency of 3' end formation, but also produced new poly(A) sites which were situated upstream of the wild-type poly(A) sites. Therefore this sequence motif in the TRP4 3'UTR acts simultaneously as both an efficiency and positioning element.

3' Untranslated Regions↗

PubMed1998

Dynamics of indigenous and inoculated yeast populations and their effect on the sensory character of Riesling and Chardonnay wines.

To study the impact of yeast populations on wine flavour and to better understand yeast growth dynamics, wines were produced by the (i) indigenous microflora, (ii) vigorous yeast starter EC1118 and (iii) slowly fermenting yeast Assmannshausen. Sensory analysis revealed that wines differed depending on the fermentation type. However, these yeast-related differences did not exceed the varietal character. Both added starter cultures clearly dominated the Saccharomyces population from the middle of fermentation onwards. The starter cultures differed in their repression of indigenous non-Saccharomyces yeast. EC1118 limited growth of non-Saccharomyces yeasts more strongly than Assmannshausen. Sulphite addition further repressed growth of non-Saccharomyces yeasts. On completion, more than one Saccharomyces strain was present in each fermentation, with the largest variety in the non-inoculated and the smallest in the EC1118-inoculated fermentation. Results from the two genetic assays, karyotyping, and PCR using delta-primers were not fully equivalent, limiting the usefulness of delta-PCR in studies of native Saccharomyces yeasts.

Antibiosis↗

PubMed1997

Sequence requirements of the bidirectional yeast TRP4 mRNA 3'-end formation signal.

The yeast TRP4 3'-end formation signal functions in both orientations in an in vivo test system. We show here that the TRP4 3'-end formation element consists of two functionally different sequence regions. One region of approximately 70 nucleotides is located in the untranslated region between the translational stop codon and the major poly(A) site. The major poly(A) site is not part of this region and can be deleted without a decrease in TRP4 3'-end formation. 5'and 3'deletions and point mutations within this region affected 3'-end formation similarly in both orientations. In the center of this region the motif TAGT is located on the antisense strand. Point mutations within this motif resulted in a drastic reduce of 3'-end formation activity in both orientations. A second region consists of the 3'-end of the TRP4 open reading frame and is required for 3'-end formation in forward orientation. A single point mutation in a TAGT motif of the TRP4 open reading frame abolished TRP4 mRNA 3'-end formation in forward orientation and had no effect on the reverse orientation.

Base Sequence↗

PubMed1995

A complex unidirectional signal element mediates GCN4 mRNA 3' end formation in Saccharomyces cerevisiae.

The yeast GCN4 3' element represents a class of polyadenylation sites which function unidirectionally and efficiently in test systems in vivo as well as in vitro. A complex signal element is required for polyadenylation activity with a minimal size of 116 nucleotides for the functional element. We subdivided this element into five regions (EL1 to EL5) of 16 to 26 nucleotides each. Each region was characterized by deletion analysis in an in vivo test system. Two TTTTTAT motifs are located in different regions (EL1 and EL4) upstream of the poly(A) site. The 3' end processing activity was significantly reduced when both motifs were mutated by site-directed mutagenesis and abolished when EL1 and EL4 were deleted. The major poly(A) site is located in EL5, 3 nucleotides downstream of the second TTTTTAT motif. Additional minor poly(A) sites are used in less than 10% of the mRNA 3' ends. Deletion of EL3 resulted in a changed pattern of mRNA 3' ends by increased usage of the minor poly(A) addition sites. The major poly(A) site in EL5 can be removed without loss of function when sequences upstream of EL1 are present. The tripartite TAG...TATGT...TTT sequence located downstream of EL5 is not required for function.

Base Sequence↗

PubMed1994

Uncoupling of mRNA 3' cleavage and polyadenylation by expression of a hammerhead ribozyme in yeast.

An artificial messenger RNA containing a derivative of a tobacco ringspot virus ribozyme was expressed in the bakers' yeast Saccharomyces cerevisiae. This mRNA was able to cleave itself efficiently in vivo. Using this system, the two steps of mRNA 3' processing, i.e. cleavage and the addition of a poly(A) tail, can be separated in yeast in vivo. The ribozyme-cleaved transcript was shown to be polyadenylated. The poly(A) tail length was similar to the poly(A) tail length of an endogenous yeast mRNA. Therefore, cleavage of the precursor RNA at the polyadenylation site and the addition of adenosine residues to the 5' product require independent cellular machineries in yeast and can be separately analyzed. This is in contrast to higher eukaryotes where both processes are coupled.

Base Sequence↗

PubMed1993

Messenger RNA 3'-end formation of a DNA fragment from the human c-myc 3'-end region in Saccharomyces cerevisiae.

We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae. A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast. The c-myc fragment had a partial capacity for directing mRNA 3'-end formation in yeast. The 3'-endpoints were 50-100 bp distant from the mRNA 3'-ends mapped in humans. This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3'-end formation.

Base Sequence↗

PubMed1993

Cloning, primary structure, and regulation of the HIS7 gene encoding a bifunctional glutamine amidotransferase: cyclase from Saccharomyces cerevisiae.

The Saccharomyces cerevisiae HIS7 gene was cloned by its location immediately downstream of the previously isolated and characterized ARO4 gene. The two genes have the same orientation with a distance of only 416 bp between the two open reading frames. The yeast HIS7 gene represents the first isolated eukaryotic gene encoding the enzymatic activities which catalyze the fifth and sixth step in histidine biosynthesis. The open reading frame of the HIS7 gene has a length of 1,656 bp resulting in a gene product of 552 amino acids with a calculated molecular weight of 61,082. Two findings implicate a bifunctional nature of the HIS7 gene product. First, the N-terminal and C-terminal segments of the deduced HIS7 amino acid sequence show significant homology to prokaryotic monofunctional glutamine amidotransferases and cyclases, respectively, involved in histidine biosynthesis. Second, the yeast HIS7 gene is able to suppress His auxotrophy of corresponding Escherichia coli hisH and hisF mutants. HIS7 gene expression is regulated by the general control system of amino acid biosynthesis. GCN4-dependent and GCN4-independent (basal) transcription use different initiator elements in the HIS7 promoter.

Amino Acid Sequence↗

PubMed1992

The yeast actin intron contains a cryptic promoter that can be switched on by preventing transcriptional interference.

We show that the single intron of the actin gene of the yeast Saccharomyces cerevisiae contains a cryptic promoter for transcription of the second exon. This promoter is inactive in the normal actin gene, but can be activated when the actin gene promoter is deleted. An identical activation was induced by placing efficient transcriptional terminators at position 61 of the 309 bp intron. In all cases transcripts with identical 5' ends close to the boundary of the intron and the second exon were produced. These results indicate that the cryptic promoter in the actin intron is occluded in the normal actin gene by transcriptional interference with the actin gene promoter. Transcription initiation near the intron/exon 2 boundary is enabled by protection from traversing polymerases, that initiated transcription at the upstream located actin gene promoter. A partial promoter protection using leaky terminators resulted in small amounts of transcripts initiated from the cryptic promoter. Although we do not know any function of the cryptic promoter in actin gene expression, it is tentative to speculate that the cryptic intron promoter might be a relict of a promoter that was functional earlier in evolution.

Actins↗

PubMed1992

Cloning, primary structure and regulation of the ARO4 gene, encoding the tyrosine-inhibited 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, two differently regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (DAHPS; EC 4.1.2.15) isoenzymes carry out the first step in the shikimate pathway. Mutations in both genes are necessary to cause aromatic amino acid (aa) auxotrophy, since one isoenzyme alone is sufficient to produce enough DAHP for normal growth of the cells. The phenylalanine-inhibited DAHPS is encoded by the previously isolated and characterized ARO3 gene. Here, we report the cloning and characterization of the ARO4 gene, encoding the second DAHPS, which is inhibited by tyrosine. The aa sequence of the ARO4 gene product reveals 76% similarity to the ARO3-encoded isoenzyme and 66 and 73% to the three DAHPS isoenzymes from Escherichia coli. ARO4 gene expression is regulated by the general control system of aa biosynthesis. As in the case of the ARO3 gene, a single GCN4-recognition element in the promoter is responsible for derepression of the ARO4 gene under aa starvation conditions. However, in contrast to the situation in the isogene, ARO3, GCN4 does not contribute to the basal level of ARO4 transcription under nonderepressing conditions.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed1992

Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.

Aldehyde Dehydrogenase↗

PubMed1991

Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae.

This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.

Base Sequence↗