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

1993 ◽  
Vol 13 (7) ◽  
pp. 4203-4213 ◽  
Author(s):  
T M Mittelmeier ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Microprojectile Bombardment ◽  
Nuclear Gene ◽  
State Level ◽  
Transcription Unit ◽  
Endonucleolytic Cleavage ◽  
In Vivo Analysis ◽  
Nucleotide Region

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).

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

1993 ◽  
Vol 13 (7) ◽  
pp. 4203-4213
Author(s):  
T M Mittelmeier ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Microprojectile Bombardment ◽  
Nuclear Gene ◽  
State Level ◽  
Transcription Unit ◽  
Endonucleolytic Cleavage ◽  
In Vivo Analysis ◽  
Nucleotide Region

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).

scholarly journals 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.

Genetics ◽  
1993 ◽  
Vol 135 (4) ◽  
pp. 981-991 ◽  
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome B ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
In Vivo Recombination ◽  
Mutant Strains ◽  
Precursor Rna

Abstract 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.

scholarly journals Drosophila Hormone Receptor 38 Functions in Metamorphosis: A Role in Adult Cuticle Formation

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1465-1475 ◽  
Author(s):  
T Kozlova ◽  
G V Pokholkova ◽  
G Tzertzinis ◽  
J D Sutherland ◽  
I F Zhimulev ◽  
...  
Keyword(s):  
Pupal Stage ◽  
Transcription Unit ◽  
P Element ◽  
Specific Response ◽  
Orphan Receptors ◽  
Mrna Isoforms ◽  
A Cell ◽  
In Vivo Analysis

Abstract DHR38 is a member of the steroid receptor superfamily in Drosophila homologous to the vertebrate NGFI-B-type orphan receptors. In addition to binding to specific response elements as a monomer, DHR38 interacts with the USP component of the ecdysone receptor complex in vitro, in yeast and in a cell line, suggesting that DHR38 might modulate ecdysone-triggered signals in the fly. We characterized the molecular structure and expression of the Dhr38 gene and initiated an in vivo analysis of its function(s) in development. The Dhr38 transcription unit spans more than 40 kb in length, includes four introns, and produces at least four mRNA isoforms differentially expressed in development; two of these are greatly enriched in the pupal stage and encode nested polypeptides. We characterized four alleles of Dhr38: a P-element enchancer trap line, l(2)02306, which shows exclusively epidermal staining in the late larval, pre-pupal and pupal stages, and three EMS-induced alleles. Dhr38 alleles cause localized fragility and rupturing of the adult cuticle, demonstrating that Dhr38 plays an important role in late stages of epidermal metamorphosis.

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

1997 ◽  
Vol 17 (8) ◽  
pp. 4199-4207 ◽  
Author(s):  
K A Sparks ◽  
S A Mayer ◽  
C L Dieckmann
Keyword(s):  
Mutant Strain ◽  
Cytochrome B ◽  
Nuclear Gene ◽  
State Level ◽  
Transcript Level ◽  
Unexpected Result ◽  
Coordinate Regulation ◽  
Growth Properties ◽  
Cytochrome B Mrna ◽  
Responsive Element

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.

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

Genetics ◽  
1994 ◽  
Vol 138 (3) ◽  
pp. 565-575
Author(s):  
R R Staples ◽  
C L Dieckmann
Keyword(s):  
Cytochrome B ◽  
Nuclear Gene ◽  
State Level ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Gene Products ◽  
Mitochondrial Cytochrome ◽  
Mitochondrial Cytochrome B ◽  
Cytochrome B Mrna ◽  
Bypass Mechanism

Abstract 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.

A functional analysis of 5′, intronic and promoter regions of the homeotic gene proboscipedia in Drosophila melanogaster

Development ◽  
1995 ◽  
Vol 121 (7) ◽  
pp. 2127-2141 ◽  
Author(s):  
A.M. Kapoun ◽  
T.C. Kaufman
Keyword(s):  
Promoter Sequence ◽  
Transcription Unit ◽  
Homeotic Gene ◽  
Lacz Reporter Gene ◽  
Promoter Regions ◽  
Hsp70 Promoter ◽  
In Vivo Analysis ◽  
Regulatory Dna ◽  
Labial Disc

In Drosophila, the homeotic gene proboscipedia (pb) is required for the formation of the adult mouthparts. To determine the functional significance of putative pb regulatory DNA, we have performed an in vivo analysis of sequences upstream of and within pb using a series of minigenes. Additionally, we have initiated a dissection of pb's promoter and enhancer elements using lacZ reporter gene constructs. Our results establish that a conserved region located in the second intron is essential for proper formation of the adult mouthparts. A 0.5 kb fragment from this region was shown to direct lacZ expression in a pb pattern in both embryos and third instar labial discs when combined with a 600 bp pb basal promoter sequence. A 32 bp element contained within the 0.5 kb region functions as a labial disc enhancers for pb. Surprisingly, the conserved second intron pb enhancers do not function properly with a heterologous hsp70 promoter, suggesting that promoter-specific interactions occur at the pb locus. We also found redundant and cryptic enhancers in the large introns of pb that are not required for pb function. Finally, we demonstrate that the pb transcription unit does not require sequences upstream of −98 bp to provide pb function in the labial discs. Rather, pb's upstream DNA appears to contain negative regulatory DNA required for silencing PB accumulation in inappropriate domains of third instar imaginal discs. Thus, we have defined many of pb's cis-controlling sequences to an experimentally manageable size, thereby making this an attractive system for the discovery of transacting proteins and, consequently, for elucidating the mechanisms of homeotic gene regulation.

scholarly journals PET111, a Saccharomyces cerevisiae Nuclear Gene Required for Translation of the Mitochondrial mRNA Encoding Cytochrome c Oxidase Subunit II

Genetics ◽  
1987 ◽  
Vol 115 (4) ◽  
pp. 637-647
Author(s):  
Candace G Poutre ◽  
Thomas D Fox
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
State Level ◽  
Mitochondrial Genes ◽  
Chimeric Proteins ◽  
Wild Type ◽  
Oxidase Subunit ◽  
A Site

ABSTRACT Mutations in the nuclear gene PET111 are recessive and specifically block accumulation of cytochrome c oxidase subunit II (coxII), the product of a mitochondrial gene. However, the coxII mRNA is present in pet111 mutants at a level approximately one-third that of wild type. The simplest explanation for this phenotype is that PET111 is required for translation of the coxII mRNA. The reduced steady-state level of this mRNA is probably a secondary effect, caused by increased degradation of the untranslated transcript. Mitochondrial suppressors of pet111, carried on rho- mtDNAs, bypass the requirement for PET111 in coxII translation. Three suppressors are fusions between the coxII structural gene and other mitochondrial genes, that encode chimeric proteins consisting of the N-terminal portions of other mitochondrially coded proteins fused to the coxII precursor protein. When present together with rho  + mtDNA in a heteroplasmic state, these suppressors allow coxII synthesis in pet111 mutants. Thus in wild type, the PET111 product, or something under its control, probably acts at a site coded in the proximal portion of the gene for coxII to promote translation of the mRNA. PET111 was isolated by molecular cloning and genetically mapped to a position approximately midway between rna1 and SUP8 on chromosome XIII.

scholarly journals Population Genetic Diversity of Two Marine Gobies (Gobiiformes: Gobiidae) from the North-Eastern Atlantic and the Mediterranean Sea

2020 ◽  
Vol 8 (10) ◽  
pp. 792
Author(s):  
Katarína Čekovská ◽  
Radek Šanda ◽  
Kristýna Eliášová ◽  
Marcelo Kovačić ◽  
Stamatis Zogaris ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Cytochrome B ◽  
Population Genetic ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Ancestral Population ◽  
Marine Biota ◽  
Population Genetic Diversity ◽  
Distribution Ranges ◽  
Mitochondrial Cytochrome B

Gobies (Gobiiformes: Gobiidae) are the most species-rich family of fishes in general, and the most abundant fish group in the European seas. Nonetheless, our knowledge on many aspects of their biology, including the population genetic diversity, is poor. Although barriers to gene flow are less apparent in the marine environment, the ocean is not a continuous habitat, as has been shown by studies on population genetics of various marine biota. For the first time, European marine goby species which cannot be collected by common fishery techniques were studied. The population genetic structure of two epibenthic species, Gobius geniporus and Gobius cruentatus, from seven localities across their distribution ranges was assessed, using one mitochondrial (cytochrome b) and one nuclear gene (first intron of ribosomal protein gene S7). Our results showed that there is a great diversity of haplotypes of mitochondrial gene cytochrome b in both species at all localities. Global fixation indices (FST) indicated a great differentiation of populations in both studied gobies. Our results did not show a geographic subdivision to individual populations. Instead, the data correspond with the model of migration which allow divergence and recurrent migration from the ancestral population. The estimated migration routes coincide with the main currents in the studied area. This matches well the biology of the studied species, with adults exhibiting only short-distance movements and planktonic larval stages.

scholarly journals ATP25, a New Nuclear Gene ofSaccharomyces cerevisiaeRequired for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

2008 ◽  
Vol 19 (4) ◽  
pp. 1366-1377 ◽  
Author(s):  
Xiaomei Zeng ◽  
Mario H. Barros ◽  
Theodore Shulman ◽  
Alexander Tzagoloff
Keyword(s):  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Ring Structure ◽  
Northern Analysis ◽  
Temperature Sensitive ◽  
Mitochondrial Atpase ◽  
Translation Product ◽  
Reading Frame ◽  
Inner Membrane Protein

We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of mitochondrial RNAs in an atp25 temperature-sensitive mutant confirmed that Atp25p is required for stability of the ATP9 mRNA. Atp25p is a mitochondrial inner membrane protein with a predicted mass of 70 kDa. The primary translation product of ATP25 is cleaved in vivo after residue 292 to yield a 35-kDa C-terminal polypeptide. The C-terminal half of Atp25p is sufficient to stabilize the ATP9 mRNA and restore synthesis of Atp9p. Growth on respiratory substrates, however, depends on both halves of Atp25p, indicating that the N-terminal half has another function, which we propose to be oligomerization of Atp9p into a proper size ring structure.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

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