Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae.

Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as "overflow metabolism" or "the Crabtree effect." The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.

Expression of the divergent transcription unit containing the yeast PET122 and OXA1 genes.

The nuclear PET122 gene of S. cerevisiae encodes a mitochondrial-localized protein that activates initiation of translation of the mitochondrial mRNA from the COX3 gene, which encodes subunit III of cytochrome c oxidase. The PET122 locus contains two divergent transcription units: one is involved in expression of PET122 mRNA and the mRNA for an adjacent gene OXA1, which is also required for cytochrome c oxidase biogenesis, and the other is involved in expression of an antisense RNA that is complementary to about two thirds of the PET122 mRNA and an adjacent gene YER152C of unknown function. Steady state levels of OXA1, PET122 sense and PET122 antisense RNAs were measured after growth of yeast cells under catabolite repressing or derepressing conditions, or after deletion of portions of the 5' flanking DNA of the genes. The results reported here indicate that the PET122 and OXA1 genes are unconventional in terms of the control of their transcription. Neither possesses a canonical TATA element and they exhibit no apparent need for native upstream DNA. These results raise the interesting possibility that PET122 and OXA1 transcription is controlled by downstream DNA, perhaps located within the coding regions of the respective genes.

Isolation of a Histoplasma capsulatum cDNA that complements a mitochondrial NAD(+)-isocitrate dehydrogenase subunit I-deficient mutant of Saccharomyces cerevisiae.

A cDNA library was prepared from Histoplasma capsulatum strain G-217B yeast cells and an apparently full-length cDNA for a subunit of the citric acid cycle enzyme NAD(+)-isocitrate dehydrogenase was identified by sequence analysis. Its predicted amino acid sequence is more similar to the IDH1 regulatory subunit of S. cerevisiae NAD(+)-isocitrate dehydrogenase than to the IDH2 catalytic subunit. After expression in S. cerevisiae from an S. cerevisiae promoter, it was shown to functionally complement an S. cerevisiae idh1 mutant, but not an idh2 mutant, for growth on acetate as a carbon source and for production of NAD(+)-isocitrate dehydrogenase enzyme activity. These results confirm that the H. capsulatum cDNA encodes a homologue of subunit I of the S. cerevisiae mitochondrial isocitrate dehydrogenase isozyme that functions in the citric acid cycle.

The Saccharomyces cerevisiae Pet309 protein is embedded in the mitochondrial inner membrane.

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for expression of the mitochondrial COX1 gene, which encodes subunit I of cytochrome c oxidase. In a pet309 null mutant, there is a defect both in accumulation of COX1 pre-RNA, if it contains introns, and in translation of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4031-4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p was tagged at the carboxy terminus with an epitope from the human c-myc protein. A monoclonal antibody against the c-myc epitope detected a 98-kDa protein in mitochondria of yeast cells that expressed the PET309-c-myc fusion protein from a high copy number plasmid. This protein was not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional analyses of mitochondrial subfractions demonstrated that the PET309-c-myc fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondria with a disrupted outer membrane). These results indicate that Pet309p spans the inner membrane, with domain(s) exposed to the intermembrane space side of the membrane. How Pet309p may function in concert with other gene products necessary for COX1 RNA translation or accumulation, such as Mss51p or Nam1p, respectively, is discussed.

Mitochondrial protein synthesis is not required for efficient excision of intron aI5 beta from COX1 pre-mRNA in Saccharomyces cerevisiae.

Splicing of the group I intron aI5 beta from the yeast mitochondrial COX1 transcript requires at least four proteins, encoded by the nuclear genes PET54, MRS1/PET157, SUV3 and MSS18. These proteins either act directly to facilitate intron aI5 beta excision, or indirectly in some manner. One possible indirect mode of action of these nuclear gene products is in stimulation of expression of a mitochondrial protein, such as a maturase, that is necessary for intron aI5 beta excision. To test this possibility, splicing of intron aI5 beta was examined in a rho-strain, which is incapable of mitochondrial protein synthesis. A quantitative RT-PCR assay was set up to compare levels of spliced COX1 mRNA present in three strains: a wild-type rho + strain; the rho-strain 7-49b-11, which retains the entire COX1 transcription unit; and a strain bearing a null mutation in the nuclear PET54 gene. The results showed that excision of aI5 beta occurs relatively efficiently in the rho-strain, and therefore does not require any mitochondrial-encoded proteins.

Setting standards for DNA banks: toward a model code of conduct.

As genomic research proliferates, DNA banking will become more common. In research, samples will be banked largely in an effort to find and clone genes that predispose to disease. Commercially oriented banks, those that offer services to families, may also become more common. These entities will hold sensitive information. DNA banking is not yet regulated. We argue here that new laws are not needed at this time to regulate DNA banking. We suggest an approach that relies on a professional code of conduct and draws on principles of disclosure inherent to the process used in obtaining informed consent. In addition to suggesting 12 specific recommendations for the code of conduct, we suggest that items should be included in depositor's agreements. We offer a rationale for our suggestions.

Crosstalk between nuclear and mitochondrial genomes.

This review focuses on molecular mechanisms that underlie the communication between the nuclear and mitochondrial genomes in eukaryotic cells. These genomes interact in at least two ways. First, they contribute essential subunit polypeptides to important mitochondrial proteins; second, they collaborate in the synthesis and assembly of these proteins. The first type of interaction is important for the regulation of oxidative energy production. Isoforms of the nuclear-coded subunits of cytochrome c oxidase affect the catalytic functions of its mitochondrially coded subunits. These isoforms are differentially regulated by environmental and developmental signals and probably allow tissues to adjust their energy production to different energy demands. The second type of interaction requires the bidirectional flow of information between the nucleus and the mitochondrion. Communication from the nucleus to the mitochondrion makes use of proteins that are translated in the cytosol and imported by the mitochondrion. Communication from the mitochondrion to the nucleus involves metabolic signals and one or more signal transduction pathways that function across the inner mitochondrial membrane. An understanding of both types of interaction is important for an understanding of OXPHOS diseases and aging.

The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae.

Expression of the Saccharomyces cerevisiae mitochondrial COX1 locus, which contains several introns and is co-transcribed with the downstream genes AAP1, OLI2 and ENS2, is controlled by at least 18 nuclear-encoded proteins. The PET309 gene, encoding one of these proteins, was cloned, sequenced and shown to contain an open reading frame of 965 codons. Isonuclear PET309+ and delta pet309::URA3 strains carrying mitochondrial genomes that differ in the number of COX1 introns, were generated. Analysis of RNA species from these strains demonstrated an inverse relationship between the number of introns present in the precursor RNA and the amount of COX1 and AAP1/OLI2/ENS2 RNAs accumulated in a pet309 mutant. Hence, PET309 plays a role either in transcription of intron-containing primary transcripts from the COX1-AAP1-OLI2-ENS2 transcription unit or in stabilization of primary transcripts. PET309 is also required in translation of COX1 mRNA. A mitochondrial bypass suppressor of the pet309 deletion mutation was isolated, and shown to consist of a DNA rearrangement at the COX1 locus, such that the 5' untranslated leader region (UTR) of the COB gene was fused to COX1 at nucleotide -174 of its 5' UTR. This result suggests that Pet309p acts through the COX1 5' UTR to activate initiation of translation of the COX1 coding region.

A survey of DNA diagnostic laboratories regarding DNA banking.

This article reports the findings of a survey of 148 academically based and commercial DNA diagnostic labs regarding DNA banking (defined as the storage of individual DNA samples in some form with identifiers for later retrieval). The population surveyed consisted of all laboratories listed with HELIX, a national directory of DNA diagnostic labs that includes a fairly comprehensive listing of clinical service labs as well as a large number of research labs. The survey was concerned primarily with the legal and ethical issues that the long-term storage of DNA may raise. The survey inquired into the respondents' policies and procedures concerning (1) the extent of DNA banking and of interest in developing DNA banking in academia and industry and (2) the degree to which DNA banks had developed written internal policies and/or a written depositor's agreement (a signed document defining the rights and obligations of the person from whom the sample was taken and the bank) designed to anticipate or prevent some of the ethical and legal problems that can arise from the long-term retention of DNA. Our research suggests that (1) the activity of DNA banking is growing, particularly in the academic setting, and (2) most academically based DNA banks lack written internal policies, written depositor's agreements, or other relevant documentation regarding important aspects of this activity.

Forensic DNA data banking by state crime laboratories.

This article reports the results of a survey of the responsible crime laboratories in the first 19 states with legislation establishing forensic DNA data banks. The survey inquired into the labs' policies and procedures regarding the collection, storage, and analysis of samples; the retention of samples and data; search protocols; access to samples and data by third parties; and related matters. The research suggests that (1) the number of samples collected from convicted offenders for DNA data banking has far surpassed the number that have been analyzed; (2) data banks have already been used in a small but growing number of cases, to locate suspects and to identify associations between unresolved cases; (3) crime labs currently plan to retain indefinitely the samples collected for their data banks; and (4) the nature and extent of security safeguards that crime labs have implemented for their data banks vary among states. The recently enacted DNA Identification Act (1994) will provide $40 million in federal matching grants to states for DNA analysis activities, so long as states comply with specified quality-assurance standards, submit to external proficiency testing, and limit access to DNA information. Although these additional funds should help to ease some sample backlogs, it remains unclear how labs will allocate the funds, as between analyzing samples for their data banks and testing evidence samples in cases without suspects. The DNA Identification Act provides penalties for the disclosure or obtaining of DNA data held by data banks that participate in CODIS, the FBI's evolving national network of DNA data banks, but individual crime labs must also develop stringent internal safeguards to prevent breaches of data-bank security.

Vectors with the gus reporter gene for identifying and quantitating promoter regions in Saccharomyces cerevisiae.

The Escherichia coli beta-glucuronidase-encoding gene gusA is useful as a reporter gene in a variety of organisms. In this report, we describe the development of two related vectors, pGUS1 and pGUS2, which can be used to identify and quantitate activities of the promoter regions from yeast genes. Both vectors contain several unique restriction sites upstream from gus and the yeast CYC1 transcription terminator downstream from gus. In addition, pGUS2 carries the yeast ADH1 transcriptional terminator sequence upstream from gus, in order to block read-through transcription originating in vector sequences. Both vectors were tested after cloning the well-characterized GAL1,10 promoter region from yeast. These GAL1,10-containing plasmids demonstrated appropriate regulation of the reporter in response to carbon sources. The pGUS1 and pGUS2 vectors provide a simple, reliable and extremely sensitive reporter-gene system that allows quantitative measurement of promoter activity of yeast DNA sequences. Furthermore, the presence of a terminator sequence upstream from gus in pGUS2 should facilitate analysis and quantitation of expression from weak promoters.

Stored Guthrie cards as DNA "banks".

Recently there has been much discussion about the possibility of using dried blood spots on Guthrie cards as a source of DNA for research or testing purposes. The collections of Guthrie cards stored by state newborn-screening laboratories can thus be viewed as inchoate "DNA banks." This has generated concern among some persons who are interested in preserving the privacy of medical records. This study examines the policies of state newborn-screening laboratories in the United States, regarding their retention of Guthrie cards and the degree to which they permit the sharing of those cards with various third parties. We found that although most laboratories retain their cards, if at all, for only a short time, a growing number plan to keep them for an extended period--and, in several cases, indefinitely. We also found that although most laboratories would decline to release individually identifiable blood spots from the cards to third parties without a written release or other explicit authorization, a large number would at least consider sharing anonymous cards for research purposes.

A review of state legislation on DNA forensic data banking.

Recent advances in DNA identification technology are making their way into the criminal law. States across the country are enacting legislation to create repositories for the storage both of DNA samples collected from convicted offenders and of the DNA profiles derived from them. These data banks will be used to assist in the resolution of future crimes. This study surveys existing state statues, pending legislation, and administrative regulations that govern these DNA forensic data banks. We critically analyzed these laws with respect to their treatment of the collection, storage, analysis, retrieval, and use of DNA and DNA data. We found much variation among data-banking laws and conclude that, while DNA forensic data banking carries tremendous potential for law enforcement, many states, in their rush to create data banks, have paid little attention to issues of quality control, quality assurance, and privacy. In addition, the sweep of some laws is unnecessarily broad. Legislative modifications are needed in many states to better safeguard civil liberties and individual privacy.

A survey of medical directors of life insurance companies concerning use of genetic information.

Rapid advances in our ability to test persons presymptomatically for genetic diseases have generated increasing concern that genetic information will be abused by insurance companies. Reasoning that the insurance companies may have the strongest interest in using genetic data and that the medical directors of those companies with responsibility for rating applicants would be a good source of information on the use of such data, we conducted a large survey of medical directors of North American life insurance companies. We received responses from 27 medical directors. Our results suggest that (1) few insurers perform genetic tests on applicants, but most are interested in accessing genetic test information about applicants that already exists; (2) the degree of insurers' interest in using genetic test results may depend on the face amount of the policy applied for and on the specificity and sensitivity of the test; (3) many companies employ underwriting guidelines with respect to certain genetic conditions but may not always have specific actuarial data in house to support their rating decisions; (4) a considerable degree of subjectivity is involved in most insurers' rating decisions; and (5) some of the medical directors who responded to our survey are not fully informed about certain basic principles of medical genetics.

Sequence and chromosomal localization of two PET genes required for cytochrome c oxidase assembly in Saccharomyces cerevisiae.

The nuclear genes PET117 and PET191 are required for the assembly of active cytochrome c oxidase in S. cerevisiae, yet their gene products are not subunits of the final assembled cytochrome c oxidase complex. Plasmids bearing PET117 or PET191 were isolated by their ability to complement the pet117-1 or pet191-1 mutations, respectively. By restriction mapping, subcloning, and deletion analysis of yeast DNA fragments that complement these mutations, the PET117 and PET191 genes were localized to smaller regions of DNA, which were then sequenced from both strands. The PET117 open reading frame is of 107 codons and the PET191 open reading frame is of 108 codons. Neither the PET191 nor PET117 DNA sequences have been reported previously, and the derived amino-acid sequences of the PET191 and PET117 open reading frames exhibit no significant primary amino-acid sequence similarity to other protein sequences available in the NBRF data base, or from translated Genbank sequences. By hybridization of PET117 or PET191 probes first to a chromosome blot and next to a library of physically mapped fragments of yeast genomic DNA, the map locations of the PET191 and PET117 genes were determined. PET117 is located on chromosome V near the HIS1 gene and PET191 is located on chromosome X near the CYC1 gene.

A survey of state insurance commissioners concerning genetic testing and life insurance.

Rapid advances in genetic testing have stimulated growing concern about the potential for misuse of genetic data by insurance companies, employers, and other third parties. Thus far, reports of genetically based discrimination in life insurance have been anecdotal. Reasoning that state insurance commissioners were likely to be aware of (1) the extent of current use of and interest in genetic tests by life insurers and (2) consumer complaints about insurance being denied because of genetic condition or because of genetic test results, we conducted a survey of that group. We received responses from 42 of the 51 jurisdictions. Our results suggest (1) that those who regulate the life insurance industry do not yet perceive genetic testing to pose a significant problem in how insurers rate applicants, (2) that life insurers have much legal latitude to require genetic tests, and (3) that so far few consumers have formally complained to commissioners about the use of genetic data by life insurers.

State legislative efforts to regulate use and potential misuse of genetic information.

The purpose of this study was to review existing and proposed legislation specifically intended to regulate the collection, use, and potential misuse of genetic data. The study encompasses laws relating to confidentiality, informed consent, discrimination, and related issues. It excludes from consideration legislation relating to medical records generally that may bear indirectly on genetic information. It also excludes both legislation relating to the regulation of DNA data collection for law enforcement purposes and state laws relating to the confidentiality of data collected by newborn-screening programs. While relatively few laws that explicitly regulate the treatment of genetic information have been enacted to date, a considerable amount of activity is currently underway in the nation's legislatures. Although most of the bills under consideration are not comprehensive in scope, they reflect a growing societal awareness that the uncontrolled dissemination and use of genetic data entails significant risks.

Characterization of mRNAs and coding potential of the PET54 gene from Saccharomyces cerevisiae.

The nuclear PET54 gene in yeast controls expression of two mitochondrial genes: COX1 at the level of pre-mRNA splicing and COX3 at the level of mRNA translation. Two size classes (1.6 and 1.1 kb) of transcripts that contain the PET54 coding region are produced in vivo. Relative to the majority of yeast mRNAs analyzed so far, the 5' untranslated leader region of the 1.6 kb transcript is unusually long (254 bases), while that for the major 1.1 kb transcript is unusually short (1 base). The majority of each class of PET54 mRNA was associated with polysomes in vivo. The possibility that two polypeptides are produced in vivo from the 1.1 kb PET54 mRNA was raised by the work of Sedman et al. [J. Virol. 64: 453-457, 1990], which showed that translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' end of the mRNA. However, two sensitive assays for production of a second polypeptide, which is predicted to be 22 kD, were employed and no second polypeptide was detected. Furthermore, a nonsense mutation introduced near the beginning of the PET54 open reading frame abolished both COX1 and COX3 gene expression. These results indicate that the PET54 gene encodes predominantly a single functional polypeptide that is employed for expression of both the COX1 and COX3 genes of mitochondrial DNA.

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII in assembly of remaining subunits.

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)