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C L Dieckmann

Publications and source records attributed to C L Dieckmann.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii.

The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.

Amino Acid Motifs↗
PubMed1999

Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae.

The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.

5' Untranslated Regions↗
PubMed1999

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.

Animals↗
PubMed1998

Regulation of poly(A) site choice of several yeast mRNAs.

Several yeast genes produce multiple transcripts with different 3'-ends. Of these, four genes are known to produce truncated transcripts that end within the coding sequence of longer transcripts: CBP1 , AEP2 / ATP13 , RNA14 and SIR1 . It has been shown that the level of the truncated CBP1 transcript increases during the switch to respiratory growth while that of the full-length transcript decreases. To determine whether this phenomenon is unique to CBP1 , northern analysis was used to determine whether the levels of other truncated transcripts are regulated similarly by carbon source. The levels of the shortest transcripts of AEP2 / ATP13 and RNA14 increased during respiration while the shortest SIR1 transcript remained constant. However, two longer SIR1 transcripts were regulated reciprocally by carbon source. Mapping the 3'-ends of each transcript by sequencing partial cDNA clones revealed multiple 3'-ends for each transcript. Examination of the sequences surrounding the 3'-ends of the induced transcripts failed to identify a consensus sequence but did reveal weak putative 3'-end formation signals in all of the transcripts. Similarly, no consensus sequence was found when the sequences surrounding the 3'-ends of the longest transcripts were compared, but again weak putative 3'-end formation signals were identified. These data are suggestive of carbon source regulation of alternative poly(A) site choice in yeast.

Base Sequence↗
PubMed1997

Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome b mRNA in Saccharomyces cerevisiae.

The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors. The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA. A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability. In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element. The CCG sequence within this region was shown to be necessary for stability. A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element. This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1997

Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae.

The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing). CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1. Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration. Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration. A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts. A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration. Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration. The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.

Base Sequence↗
PubMed1996

Independent regulation of full-length and 5'-truncated PAS5 mRNAs in Saccharomyces cerevisiae.

Peroxisome assembly in Saccharomyces cerevisiae requires the products of several genes. In this report, the PAS5 gene has been characterized. The gene is on the left arm of chromosome X and encodes a polypeptide with similarity to the mammalian peroxisome assembly factor-1 (PAF-1). Two different length transcripts are produced from the yeast PAS5 gene. The longer mRNAs encompass an open reading frame, while the shorter transcripts initiate 46-110 base pairs downstream of the first in-frame AUG. The longer transcripts are induced four-fold on medium containing fatty acids as the sole carbon source, while the shorter transcripts are induced up to ten-fold on medium containing glycerol as a carbon source. The full-length coding sequence encodes a protein with a calculated molecular weight of 30.7 kDa. A protein of 25 kDa could be translated from the shorter transcripts and would lack a very acidic domain found in the amino-terminal extension of the longer protein. The common portion of the proteins is very basic; the calculated pI of the longer polypeptide is 9.02 and that of the shorter protein is 10.06.

Amino Acid Sequence↗
PubMed1995

In vivo analysis of sequences required for translation of cytochrome b transcripts in yeast mitochondria.

Respiratory chain proteins encoded by the yeast mitochondrial genome are synthesized within the organelle. Mitochondrial mRNAs lack a 5' cap structure and contain long AU-rich 5' untranslated regions (UTRs) with many potential translational start sites and no apparent Shine-Dalgarno-like complementarity to the 15S mitochondrial rRNA. However, translation initiation requires specific interactions between the 5' UTRs of the mRNAs, mRNA-specific activators, and the ribosomes. In an initial step toward identifying potential binding sites for the mRNA-specific translational activators and the ribosomes, we have analyzed the effects of deletions in the 5' UTR of the mitochondrial COB gene on translation of COB transcripts in vivo. The deletions define two regions of the COB 5' UTR that are important for translation and indicate that sequence just 5' of the AUG is involved in selection of the correct start codon. Taken together, the data implicate specific regions of the 5' UTR of COB mRNA as possible targets for the mitochondrial translational machinery.

Base Sequence↗
PubMed1994

Cbp1p is required for message stability following 5'-processing of COB mRNA.

Cbp1p is a nuclear encoded protein required for the stabilization of mitochondrial COB mRNA, which codes for apocytochrome b in the yeast Saccharomyces cerevisiae. The COB gene is cotranscribed with the upstream tRNA(Glu) gene. Release of tRNA(Glu) from the initial transcript generates a precursor mRNA with a 5'-end at position -1098. The 5'-end of mature COB message is generated by cleavage of the pre-mRNA at nucleotide -955 or -954. Previous work indicated that Cbp1p acts through cis-elements near these cleavage sites. Here we have tested whether Cbp1p stabilizes COB mRNA solely by stimulating the processing of COB precursor RNA at nucleotide -955/-954. Yeast strain TG955 was constructed such that the -955 COB mRNA 5'-processing site coincides with the upstream tRNA 3'-endonuclease site at position -1098, allowing the 5'-end of COB mRNA to be formed by the tRNA 3'-processing enzyme. Respiratory growth and stability of COB mRNA in TG955 are Cbp1p-dependent. Therefore, we conclude that Cbp1p is important for the stabilization of COB mRNA after 5'-processing.

Apoproteins↗
PubMed1994

Suppressor analyses of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae: the product of the nuclear gene SOC1 affects mitochondrial cytochrome b mRNA post-transcriptionally.

The induction of mitochondrial function is dependent upon both nuclearly encoded and mitochondrially encoded gene products. To understand nuclear-mitochondrial interactions, we must first understand gene-specific interactions. The accumulation of mitochondrial cytochrome b (COB) RNA is dependent upon Cbp1p, encoded by the nuclear gene CBP1. Thus, respiration is dependent upon Cbp1p. In this study, suppressors of temperature-sensitive cbp1 (cbp1ts) strains were selected for restoration of respiratory capability at the restrictive temperature Ts+). One nuclearly encoded suppressor, extragenic to CBP1, is recessive with respect to the wild-type suppressor allele and is unlinked to other known genetic loci whose gene products are necessary for expression of COB mRNA. The suppressor, called soc1 for Suppressor of cbp1, suppresses several other cbp1ts alleles but does not operate via a bypass mechanism. Molecular analyses indicate that soc1 allows the steady-state level of COB mRNA to increase at high temperature but has little or no effect on the levels of COB pre-mRNA. These data have led us to propose that the product of the nuclear gene SOC1 is required for normal turnover of COB mRNA.

Base Sequence↗
PubMed1993

Generation of temperature-sensitive cbp1 strains of Saccharomyces cerevisiae by PCR mutagenesis and in vivo recombination: characteristics of the mutant strains imply that CBP1 is involved in stabilization and processing of cytochrome b pre-mRNA.

Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBP1 of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBP1 protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBP1, we generated temperature-sensitive cbp1 mutant strains by polymerase chain reaction (PCR) mutagenesis and in vivo recombination. These temperature-sensitive cbp1 strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBP1 protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA.

Base Sequence↗
PubMed1993

In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria.

In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).

Base Sequence↗
PubMed1991

Yeast CBP1 mRNA 3' end formation is regulated during the induction of mitochondrial function.

Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene. While many mammalian genes contain multiple mRNA 3' cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity. The Saccharomyces cerevisiae nuclear gene CPB1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability. This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CPB1. Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3' end near the middle of the coding sequence. Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA. Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3' end formation of the 1.2-kb transcript, a unique case in yeast cells.

Base Sequence↗
PubMed1990

Identification of the CBP1 polypeptide in mitochondrial extracts from Saccharomyces cerevisiae.

CBP1 is a nuclearly encoded yeast protein required for stability of mitochondrial cytochrome b pre-mRNA. Previous studies have shown that CBP1 stabilizes the cytochrome b transcripts via interaction with the 5'-end. For the present study, both rabbit polyclonal and mouse monoclonal antibodies against CBP1 were prepared using a trpE-CBP1 fusion polypeptide as a source of antigen. CBP1 was undetectable in a crude mitochondrial fraction from a wild-type strain by Western blot assay, but a 66-kDa immunoreactive protein was detected in a more purified fraction. The 66-kDa protein was absent in the equivalent fraction from a strain with a deletion in CBP1. Assignment of Mr = 66,000 to the mature CBP1 polypeptide was verified by Western analysis of mitochondria from a strain which over-expresses CBP1. Mitochondrial localization was verified by transcribing CBP1 in vitro with T3 polymerase, translating the artificial mRNA in a rabbit reticulocyte system and importing 35S-CBP1 precursor polypeptides into isolated mitochondria. The mature protein product was 66 kDa, whereas the precursor protein migrated as if it were 68 kDa rather than 76 kDa as predicted from the sequence. Analysis of polypeptides truncated at the carboxyl terminus showed that CBP1 polypeptides migrate anomalously fast in the Laemmli system due to a property of the carboxyl two-thirds of the primary sequence, several sections of which are extremely basic.

Antigens, Fungal↗
PubMed1990

CBP1 function is required for stability of a hybrid cob-oli1 transcript in yeast mitochondria.

The nuclear gene product CBP1 stabilizes cytochrome b transcripts in yeast mitochondria. In cbp1 mutant strains, cytochrome b gene (cob) transcripts are not detectable by Northern blot analysis. The results of previous studies led to the hypothesis that CBP1 interacts with the 5'-untranslated sequence of the cob mRNA, or pre-mRNA, to stabilize the message. To determine what portion of the cob leader is sufficient for interaction with CBP1, we have investigated the stability of transcripts from a novel hybrid gene, cob-oli1, in which the 5'-terminal third of the cob leader sequence was fused to the coding sequence of the gene for ATP synthase subunit 9, oli1. The hybrid cob-oli1 transcript was stable in a strain wild-type at the CBP1 locus, but was undetectable in the cbp1 mutant background. That the cob-oli1 transcript was translated to produce ATP synthase subunit 9 in CBP1 strains containing the cob-oli1 gene was verified by 35S-methionine labeling of mitochondrial proteins. We conclude that the 5'-terminal portion of the cob message is sufficient for CBP1 function and discuss the hypothesis that CBP1 interacts directly with this region of the transcript to promote cob mRNA stability.

Base Sequence↗
PubMed1990

PET genes of Saccharomyces cerevisiae.

We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae.

Genes, Fungal↗
PubMed1989

The yeast CBP1 gene produces two differentially regulated transcripts by alternative 3'-end formation.

CBP1 is a yeast nuclear gene encoding a mitochondrial protein that stabilizes the 5' end of cytochrome b (cob) pre-mRNA. Cytochrome b is the only mitochondrially synthesized component of the respiratory chain complex III. Since the nuclearly encoded subunits of this complex are regulated at the transcriptional level by catabolite repression, we hypothesized that CBP1 might be similarly regulated. To test the idea that transcriptional regulation of CBP1 could coordinate an increase in cytochrome b mRNA stability with an increase in nuclearly encoded complex III subunit production, we characterized the change in abundance of CBP1 mRNA during derepression on a nonfermentable carbon source. Poly(A)+ RNA from derepressed yeast cells was examined by Northern (RNA) analyses with cRNA probes from CBP1. Both 2.2- and 1.3-kilobase (kb) transcripts were detected. The 1.3-kb mRNA lacked approximately 900 nucleotides of the 3' end of the 2.2-kb mRNA, which encodes the carboxyl-terminal 250 amino acid residues of the CBP1 coding sequence. Northern analyses of RNA isolated from deletion-insertion mutants of CBP1 and from strains that overexpress CBP1 mRNA demonstrated that both mRNAs were transcribed from the CBP1 gene. Furthermore, we demonstrated that the levels of the two CBP1 mRNAs were reciprocally regulated by the carbon source in the growth medium. This is the first description of a yeast gene from which two transcripts that can encode proteins with distinctly different coding properties are generated by alternative 3'-end formation.

Blotting, Northern↗