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Two Differently Regulated mRNAs with Different 5' Ends Encode Secreted and Intracellular Forms of Yeast Invertase
Abstract
The SUC2 gene of yeast (Saccharomyces) encodes two forms of invertase: a secreted, glycosylated form, the synthesis of which is regulated by glucose repression, and an intracellular, nonglycosylated enzyme that is produced constitutively. The SUC2 gene has been cloned and shown to encode two RNAs (1.8 and 1.9 kb) that differ at their 5' ends. The stable level of the larger RNA is regulated by glucose; the level of the smaller RNA is not. A correspondence between the presence of the 1.9 kb RNA and the secreted invertase, and between the 1.8 kb RNA and the intracellular invertase, was observed in glucose-repressed and -derepressed wild-type cells. In addition, cells carrying a mutation at the SNF1 locus fail to derepress synthesis of the secreted invertase and also fail to produce stable 1.9 kb RNA during growth in low glucose. Glucose regulation of invertase synthesis thus is exerted, at least in part, at the RNA level. A naturally silent allele (suc2 degrees) of the SUC2 locus that does not direct the synthesis of active invertase was found to produce both the 1.8 and 1.9 kb RNAs under normal regulation by glucose. A model is proposed to account for the synthesis and regulation of the two forms of invertase: the larger, regulated mRNA contains the initiation codon for the signal sequence required for synthesis of the secreted, glycosylated form of invertase; the smaller, constitutively transcribed mRNA begins within the coding region of the signal sequence, resulting in synthesis of the intracellular enzyme.
... In contrast to maltose which, as described above, is taken up by proton symport prior to hydrolysis [31,32,42], sucrose metabolism in wild-type S. cerevisiae strains is predominantly initiated by its extracellular hydrolysis to glucose and fructose, catalysed by invertase (Suc2, EC 3.2.1.26) (Fig. 1) [65,66]. After uptake via facilitated diffusion, mediated by Hxt transporters [67], these hexoses are oxidized to pyruvate by yeast glycolysis. ...
... Due to the presence of a second start codon in the SUC2 transcript, a small fraction of the expressed invertase is retained in the cytosol [66] while, moreover, the Mal11 (Agt1) maltose-proton symporter is also able to import sucrose [68,69]. Replacement of the native SUC2 gene by a constitutively expressed, truncated SUC2 gene that no longer encoded the N-terminal excretion sequence of Suc2 led to a near-complete targeting of invertase to the yeast cytosol ( Fig. 2G) [70]. ...
Product yield on carbohydrate feedstocks is a key performance indicator for industrial ethanol production with the yeast Saccharomyces cerevisiae. This paper reviews pathway engineering strategies for improving ethanol yield on glucose and/or sucrose in anaerobic cultures of this yeast by altering the ratio of ethanol production, yeast growth and glycerol formation. Particular attention is paid to strategies aimed at altering energy coupling of alcoholic fermentation and to strategies for altering redox-cofactor coupling in carbon and nitrogen metabolism that aim to reduce or eliminate the role of glycerol formation in anaerobic redox metabolism. In addition to providing an overview of scientific advances we discuss context dependency, theoretical impact and potential for industrial application of different proposed and developed strategies.
... Le saccharose est constitué d'une unité de glucose liée en β-1,2 à une unité de fructose. Dans la plupart des cas, son hydrolyse est effectuée par une invertase (β-Dfructofuranosidase) localisée dans l'espace périplasmique dont l'expression est régulée par la concentration du glucose (Carlson and Botstein, 1982 ;Özcan et al., 1997 ;Flores et al., 2000). Cependant, une forme cytoplasmique de l'invertase a également été rapportée chez certaines levures telles que S. cerevisiae et Candida albicans (Stambuk et al., 1999 ;Flores et al., 2000). ...
La biomasse lignocellulosique (BLC), composée des déchets végétaux agricoles et/ou forestiers, représente une ressource renouvelable prometteuse pour la production de bioéthanol de deuxième génération et faire face à l'épuisement des énergies fossiles. Le procédé de valorisation de la BLC appliqué ici comprend une étape de prétraitement par deux liquides ioniques (LI) hydrophiles dérivés d'imidazolium, le 1-éthyl-3-méthylimidazolium acétate [Emim][OAc] et le 1-éthyl-3-méthylimidazolium méthylphosphonate [Emim][MeO(H)PO2], puis une hydrolyse enzymatique par des cellulases de Trichoderma reesei et des xylanases de Trichoderma longibrachiatum, suivie d'une étape de fermentation éthanolique microbienne testée sur quatre espèces de levures: Saccharomyces cerevisiae, Kluyveromyces marxianus, Scheffersomyces shehatae et Scheffersomyces stipitis. Deux BLC réelles ont été étudiées : le saule blanc (résidu forestier) et le son de blé désamidonné (résidu agricole). Les résultats ont montré que le prétraitement des BLC aux LI à 35°C permet une augmentation de la digestibilité enzymatique des polysaccharides (rendements en glucose jusqu'à +56,7% et rendements en xylose jusqu'à +31,1%) par rapport au contrôle sans prétraitement. De plus, la performance des procédés de fermentation éthanolique est améliorée d'un facteur 1,3 à 4,6 en fonction du substrat étudié et de l'espèce de levure utilisée. Ainsi, les LI sont des solvants prometteurs pour le prétraitement de la (ligno)cellulose à basse température permettant une hausse des rendements d'hydrolyse enzymatique et de production éthanolique
... The vast majority (96%, n = 815) was specific either to doubling time or yield, consistent with the lack of correlation between these fitness components (mean Pearson's r 2 = 0. 16) and implying that they are genetically independent and probably evolved separately. We recovered known causes of domestication effects, with gain of SUC2 or DAL5 or amplification of MAL genes, explaining better growth on sucrose, citrulline and maltose, respectively [38][39][40] . We also disclosed few unknown variants with extensive pleiotropic effects. ...
Domestication of plants and animals is the foundation for feeding the world human population but can profoundly alter the biology of the domesticated species. Here we investigated the effect of domestication on one of our prime model organisms, the yeast Saccharomyces cerevisiae, at a species-wide level. We tracked the capacity for sexual and asexual reproduction and the chronological life span across a global collection of 1,011 genome-sequenced yeast isolates and found a remarkable dichotomy between domesticated and wild strains. Domestication had systematically enhanced fermentative and reduced respiratory asexual growth, altered the tolerance to many stresses and abolished or impaired the sexual life cycle. The chronological life span remained largely unaffected by domestication and was instead dictated by clade-specific evolution. We traced the genetic origins of the yeast domestication syndrome using genome-wide association analysis and genetic engineering and disclosed causative effects of aneuploidy, gene presence/absence variations, copy number variations and single-nucleotide polymorphisms. Overall, we propose domestication to be the most dramatic event in budding yeast evolution, raising questions about how much domestication has distorted our understanding of the natural biology of this key model species. This study assesses the capacity for sexual and asexual reproduction and the chronological life span across 1,011 genome-sequenced budding yeast isolates and shows the remarkable impact of domestication on budding yeast evolution.
... Analysis of DEGs in Glycolysis/Gluconeogensis (ko00010), Fructose and Mannose Metabolism (ko00051), Galactose Metabolism (ko00052), Starch and Sucrose metabolism (ko00500), Pentose Phosphate Pathway (ko00030), Citrate cycle (ko00020), and Pentose and Glucuronate interconversions (ko00040) in Gcn5 and Hos2 revealed that the number of down-regulated genes ( Gcn5, 22; Hos2, 15) was significantly greater than the up-regulated genes ( Gcn5, 22; Hos2, 15) (Figure 7). In yeast, invertase and hexokinase are critical for the utilization of sucrose, lactose, glucose, or fructose (Carlson and Botstein, 1982). In A. alternata, AALT_g6039 encodes hexokinase, and AALT_g5797, AALT_g10082 and AALT_g2401 encode invertases. ...
Histone acetylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by HATs and HDACs. This study determined the function of 6 histone acetyltransferases (HATs) ( Gcn5 , RTT109 , Elp3 , Sas3 , Sas2 , Nat3 ) and 6 histone deacetylases (HDACs) ( Hos2 , Rpd3 , Hda1 , Hos3 , Hst2 , Sir2 ) in the phytopathogenic fungus Alternaria alternata by analyzing targeted gene deletion mutants. Our data provide evidence that HATs and HDACs are both required for mycelium growth, cell development and pathogenicity as many gene deletion mutants (Δ Gcn5 , Δ RTT109 , Δ Elp3 , Δ Sas3 , Δ Nat3 , Δ Hos2 , and Δ Rpd3 ) displayed reduced growth, conidiation or virulence at varying degrees. In addition, HATs and HDACs are involved in the resistance to multiple stresses such as oxidative stress ( Sas3 , Gcn5 , Elp3 , RTT109 , Hos2 ), osmotic stress ( Sas3 , Gcn5 , RTT109 , Hos2 ), cell wall-targeting agents ( Sas3 , Gcn5 , Hos2 ), and fungicide ( Gcn5 , Hos2 ). Δ Gcn5 , Δ Sas3 , and Δ Hos2 displayed severe growth defects on sole carbon source medium suggesting a vital role of HATs and HDACs in carbon source utilization. More SNPs were generated in Δ Gcn5 in comparison to wild-type when they were exposed to ultraviolet ray. Moreover, Δ RTT109 , Δ Gcn5 , and Δ Hos2 showed severe defects in resistance to DNA-damaging agents, indicating the critical role of HATs and HDACs in DNA damage repair. These phenotypes correlated well with the differentially expressed genes in Δ Gcn5 and Δ Hos2 that are essential for carbon sources metabolism, DNA damage repair, ROS detoxification, and asexual development. Furthermore, Gcn5 is required for the acetylation of H3K4. Overall, our study provides genetic evidence to define the central role of HATs and HDACs in the pathological and biological functions of A. alternata .
... The strain used in this study (S288C) carries a gene that encodes only one functional invertase enzyme, SUC2 (12,15). There is a constitutively expressed intracellular form of invertase, but the secreted, glycosylated form which is regulated by glucose repression and important for cooperativity is secreted into the periplasmic space (16). Most invertase (95%) remains in the cell wall; nevertheless, yeast capture only a small fraction of the sugars that sucrose hydrolysis releases with most of the glucose and fructose diffusing away to be utilized by other cells (17)(18)(19). ...
Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions.
... Clb2p accumulation showed an extended delay in YEP-GAL (Fig. 3B, 180 min) compared with YEPD media (Fig. 3A, 60-80 min). This extended delay in cell-cycle progression might result from glucose repression that involves the transcriptional repression of genes (GAL genes and many other genes) that metabolize nonpreferred carbon sources (Carlson and Botstein, 1982;Nehlin et al., 1991;Wilson et al., 1996;De Vit et al., 1997). To examine fMAPK pathway activity in response to a sustained induction, cells pregrown in YEP-GAL medium were synchronized and monitored. ...
Mitogen-Activated Protein Kinase (MAPK) pathways control cell differentiation and the response to stress. In Saccharomyces cerevisiae, the MAPK pathway that controls filamentous growth (fMAPK) shares components with the pathway that regulates the response to osmotic stress (HOG). Here, we show that the two pathways exhibit different patterns of activity throughout the cell cycle. The different patterns resulted from different expression profiles of genes encoding mucin sensors that regulate the pathways. Cross-pathway regulation from the fMAPK pathway stimulated the HOG pathway, presumably to modulate fMAPK pathway activity. We also show that the shared tetraspan protein, Sho1p, which has a dynamic localization pattern, induced the fMAPK pathway at the mother-bud neck. A Sho1p-interacting protein, Hof1p, which also localizes to the mother-bud neck and regulates cytokinesis, also regulated the fMAPK pathway. Therefore, spatial and temporal regulation of pathway sensors, and cross-pathway regulation, control a MAPK pathway that regulates cell differentiation in yeast.
... However, the yeast strain used for these assays was also the EBYVW.4000 which contains the SUC2 gene, coding for secreted and intracellular invertase (Carlson and Botstein, 1982). Therefore, the possibility that sucrose might be cleaved and taken up exists, and thus another strain with a deletion in SUC2 could help to rule out that possibility (Helber et al., 2011). ...
Root colonization by filamentous fungi modifies sugar partitioning in plants by increasing the sink strength. As a result, a transcriptional reprogramming of sugar transporters takes place. Here we have further advanced in the characterization of the potato SWEET sugar transporters and their regulation in response to the colonization by symbiotic and pathogenic fungi. We previously showed that root colonization by the AM fungus Rhizophagus irregularis induces a major transcriptional reprogramming of the 35 potato SWEETs, with 12 genes induced and 10 repressed. In contrast, here we show that during the early colonization phase, the necrotrophic fungus Fusarium solani only induces one SWEET transporter, StSWEET7a , while represses most of the others (25). StSWEET7a was also induced during root colonization by the hemi-biotrophic fungus Fusarium oxysporum f. sp. tuberosi . StSWEET7a which belongs to the clade II of SWEET transporters localized to the plasma membrane and transports glucose, fructose and mannose. Overexpression of StSWEET7a in potato roots increased the strength of this sink as evidenced by an increase in the expression of the cell wall-bound invertase. Concomitantly, plants expressing StSWEET7a were faster colonized by R. irregularis and by F. oxysporum f. sp. tuberosi . The increase in sink strength induced by ectopic expression of StSWEET7a in roots could be abolished by shoot excision which reverted also the increased colonization levels by the symbiotic fungus. Altogether, these results suggest that AM fungi and Fusarium spp. might induce StSWEET7a to increase the sink strength and thus this gene might represent a common susceptibility target for root colonizing fungi.
... Using N-terminal proteomics approaches, several groups have reported that translation start at an in-frame downstream initiation codon is a commonly observed trait in yeast [42][43][44]. While the biological role of many of these proteoforms remains elusive, several authors have showed that selection of appropriate transcription and translation start sites might control the subcellular localization of the resulting isoforms [45][46][47][48][49][50]. Our study shows that Ccc1 localizes in the vacuolar membrane, whereas s-Ccc1 localizes both in the vacuolar and ER membranes ( Figure 6). ...
In yeast, iron storage and detoxification depend on the Ccc1 transporter that mediates iron accumulation in vacuoles. While deletion of the CCC1 gene renders cells unable to survive under iron overload conditions, the deletion of its previously identified regulators only partially affects survival, indicating that the mechanisms controlling iron storage and detoxification in yeast are still far from well understood. This work reveals that CCC1 is equipped with a complex transcriptional structure comprising several regulatory regions. One of these is located inside the coding sequence of the gene and drives the expression of a short transcript encoding an N-terminally truncated protein, designated as s-Ccc1. s-Ccc1, though less efficiently than Ccc1, is able to promote metal accumulation in the vacuole, protecting cells against iron toxicity. While the expression of the s-Ccc1 appears to be repressed in the normal genomic context, our current data clearly demonstrates that it is functional and has the capacity to play a role under iron overload conditions.
The GLN3 gene of Saccharomyces cerevisiae is required for the activation of transcription of a number of genes in response to the replacement of glutamine by glutamate as source of nitrogen. We cloned the GLN3 gene and constructed null alleles by gene disruption. GLN3 is not essential for growth, but increased copies of GLN3 lead to a drastic decrease in growth rate. The complete nucleotide sequence of the GLN3 gene was determined, revealing one open reading frame encoding a polypeptide of 730 amino acids, with a molecular weight of approximately 80,000. The GLN3 protein contains a single putative Cys2/Cys2 zinc finger which has homology to the Neurospora crassa NIT2 protein, the Aspergillus nidulans AREA protein, and the erythroid-specific transcription factor GATA-1. Immunoprecipitation experiments indicated that the GLN3 protein binds the nitrogen upstream activation sequence of GLN1, the gene encoding glutamine synthetase. Neither control of transcription nor control of initiation of translation of GLN3 is important for regulation in response to glutamine availability.
The SNF1 gene of Saccharomyces cerevisiae is essential for normal regulation of gene expression by glucose repression. A functional SNF1 gene product is required to derepress many glucose-repressible genes in response to conditions of low external glucose. In the case of the SUC2 structural gene for invertase, SNF1 acts at the RNA level. We have reported the isolation of a cloned gene that complements the snf1 defect in S. cerevisiae and that is homologous to DNA at the SNF1 locus (J. L. Celenza and M. Carlson, Mol. Cell. Biol. 4:49-53, 1984). In this work we identified a 2.4-kilobase polyadenylate-containing RNA encoded by the SNF1 gene and showed that its level is neither regulated by glucose repression nor dependent on a functional SNF1 product. The position of the SNF1 RNA relative to the cloned DNA was mapped, and the direction of transcription was determined. The cloned DNA was used to disrupt the SNF1 gene at its chromosomal locus. Gene disruption resulted in A Snf1- phenotype, thereby proving that the cloned gene is the SNF1 gene and showing that the phenotype of a true null mutation is indistinguishable from that of previously isolated snf1 mutations.
We have cloned the negative regulatory gene (DAL80) of the allantoin catabolic pathway, characterized its structure, and determined the physiological conditions that control DAL80 expression and its influence on the expression of nitrogen catabolic genes. Disruption of the DAL80 gene demonstrated that it regulates multiple nitrogen catabolic pathways. Inducer-independent expression was observed for the allantoin pathway genes DAL7 and DUR1,2, as well as the UGA1 gene required for gamma-aminobutyrate catabolism in the disruption mutant. DAL80 transcription was itself highly sensitive to nitrogen catabolite repression (NCR), and its promoter contained 12 sequences homologous to the NCR-sensitive UASNTR. The deduced DAL80 protein structure contains zinc finger and coiled-coil motifs. The DAL80 zinc finger motif possessed high homology to the transcriptional activator proteins required for expression of NCR-sensitive genes in fungi and the yeast GLN3 gene product required for functioning of the NCR-sensitive DAL UASNTR. It was also homologous to the three GATAA-binding proteins reported to be transcriptional activators in avian and mammalian tissues. The latter correlations raise the possibility that both positive and negative regulators of allantoin pathway transcription may bind to similar sequences.
The GAL4 gene encodes a positive regulator of the galactose-inducible genes in Saccharomyces cerevisiae. Recently, GAL4 has been cloned and its 2.8-kilobase mRNA has been identified. We report here the DNA sequence of GAL4 and the mapping of the 5' and 3' ends of its transcripts. The region sequenced contains a single open reading frame, 881 codons long, which could encode a 99,350-dalton protein. The 5' ends of the GAL4 transcripts fall into two clusters. Transcripts which begin at the upstream cluster would encode the 99,350-dalton protein, whereas those starting at the downstream cluster may result in the synthesis of a shorter, 91,600-dalton protein. The putative GAL4 proteins contain an amino acid sequence near their amino termini which resembles a DNA-binding motif found in bacterial and phage repressors and gene activator proteins.
SPT16 was previously identified as a high-copy-number suppressor of delta insertion mutations in the 5' regions of the HIS4 and LYS2 genes of Saccharomyces cerevisiae. We have constructed null mutations in the SPT16 gene and have demonstrated that it is essential for growth. Temperature-sensitive-lethality spt16 alleles have been isolated and shown to be pleiotropic; at a temperature permissive for growth, spt16 mutations suppress delta insertion mutations, a deletion of the SUC2 upstream activating sequence, and mutations in trans-acting genes required for both SUC2 and Ty expression. In addition, SPT16 is identical to CDC68, a gene previously shown to be required for passage through the cell cycle control point START. However, at least some transcriptional effects caused by spt16 mutations are independent of arrest at START. These results and those in the accompanying paper (A. Rowley, R. A. Singer, and G. C. Johnston, Mol. Cell. Biol. 11:5718-5726, 1991) indicate that SPT16/CDC68 is required for normal transcription of many loci in S. cerevisiae.
The MEL1 gene in Saccharomyces cerevisiae is required for the production of alpha-galactosidase and for the catabolism of melibiose. Production of alpha-galactosidase is induced by galactose or melibiose and repressed by glucose. Inducibility is controlled by the positive and negative regulatory proteins GAL4 and GAL80, respectively. We have cloned the MEL1 gene to study its transcriptional expression and regulation. Evidence is presented that the MEL1 gene encodes alpha-galactosidase and that mel0 is a naturally occurring allele which lacks the alpha-galactosidase-coding sequences. RNAs prepared from wild-type cells and from cells carrying either the noninducible gal4-2 or GAL80S-100 allele grown on three different carbon sources were examined by Northern hybridization analyses. In wild-type cells under noninducing conditions, such as growth on glycerol-lactic acid, the MEL1 transcript was detected at a basal level which was 1 to 2% of the fully induced level. The basal level of expression was diminished in cells carrying the gal4-2 mutant allele but not in cells carrying the GAL80S-100 allele. The basal and induced RNA levels are repressed by glucose. Size determinations of the MEL1 transcripts detected in glycerol-lactic acid- and galactose-grown cells provided no evidence for two distinct transcripts.
A functional SNF1 gene product is required to derepress expression of many glucose-repressible genes in Saccharomyces cerevisiae. Strains carrying a snf1 mutation are unable to grow on sucrose, galactose, maltose, melibiose, or nonfermentable carbon sources; utilization of these carbon sources is regulated by glucose repression. The inability of snf1 mutants to utilize sucrose results from failure to derepress expression of the structural gene for invertase at the RNA level. We isolated recombinant plasmids carrying the SNF1 gene by complementation of the snf1 defect in S. cerevisiae. A 3.5-kilobase region is common to the DNA segments cloned in five different plasmids. Transformation of S. cerevisiae with an integrating vector carrying a segment of the cloned DNA resulted in integration of the plasmid at the SNF1 locus. This result indicates that the cloned DNA is homologous to sequences at the SNF1 locus. By mapping a plasmid marker linked to SNF1 in this transformant, we showed that the SNF1 gene is located on chromosome IV. We then mapped snf1 to a position 5.6 centimorgans distal to rna3 on the right arm; snf1 is not extremely closely linked to any previously mapped mutation.
Microscopic screening of a collection of cold-sensitive mutants of Saccharomyces cerevisiae led to the identification of a new gene, CDC55, which appears to be involved in the morphogenetic events of the cell cycle. CDC55 maps between CDC43 and CHC1 on the left arm of chromosome VII. At restrictive temperature, the original cdc55 mutant produces abnormally elongated buds and displays a delay or partial block of septation and/or cell separation. A cdc55 deletion mutant displays a cold-sensitive phenotype like that of the original isolate. Sequencing of CDC55 revealed that it encodes a protein of about 60 kDa, as confirmed by Western immunoblots using Cdc55p-specific antibodies. This protein has greater than 50% sequence identity to the B subunits of rabbit skeletal muscle type 2A protein phosphatase; the latter sequences were obtained by analysis of peptides derived from the purified protein, a polymerase chain reaction product, and cDNA clones. An extragenic suppressor of the cdc55 mutation lies in BEM2, a gene previously identified on the basis of an apparent role in bud emergence.
A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity of rad2-1, rad2-2, and rad2-4 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomic library. Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmid containing a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at the chromosomal rad2 gene. We therefore conclude that this plasmid contains the RAD2 gene. The 9.3-kilobase pair fragment was partially digested with Sau3A and cloned into a multicopy yeast vector designed for easy retrieval of Sau3A inserts. The smallest subclone that retains the RAD2 gene is 4.5 kilobase pairs. This fragment was partially digested with Sau3A and cloned into an integrating plasmid. These plasmids were isolated and integrated into a heterozygous rad2/RAD2 strain. Plasmids containing internal fragments of the RAD2 gene were identified because they yielded UV-sensitive transformants due to disruption of the RAD2 gene. Sporulation of diploids transformed with integrating plasmids containing internal fragments of RAD2 gave rise to four viable haploids per tetrad, indicating that unlike the RAD3 gene of S. cerevisiae, the RAD2 gene is not essential for the viability of haploid cells under normal growth conditions. Measurements of the RNA transcript by RNA-DNA hybridization with the internal fragment as the probe indicate a size of approximately 3.2 kilobases.
We have isolated three cis-dominant mutations which dramatically enhance DUR1 ,2 gene expression in Saccharomyces cerevisiae. The mutant phenotype, which is expressed both in haploid and MATa/MAT alpha diploid strains, does not appear to be an alteration of the normal control system for this gene because its expression remained fully inducible and sensitive to nitrogen catabolite repression. Instead, we found much higher levels of DUR1 ,2-specific RNA under both uninduced and induced conditions, i.e., the overproduction trait was superimposed on normal regulation of the gene. The mutations seemed to affect gene expression in a unidirectional manner or to be specific for DUR1 ,2 gene expression, because other genes in proximity to the mutations were not affected. We feel that these mutations may alter the chromatin structure in the vicinity of the DUR1 ,2 upstream control sequences or, alternatively, may be Ty insertions which no longer possess the ROAM characteristics reported by others and ourselves.
The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.
A single structural gene, SUC2, encodes both secreted and cytoplasmic invertase in Saccharomyces cerevisiae. It is known that the unprocessed polypeptides which differ by a secretion signal sequence are encoded by separate mRNAs. This unusual transcriptional organization raises the question as to the degree to which the transcripts can be independently regulated. To define a system for studying this problem, we examined invertase transcription after various physiological perturbations of cells: rapid catabolite derepression, heat shock, and cell cycle arrest. With each treatment, fluctuations in mRNA levels for both cytoplasmic and secreted invertase were observed. We concluded that (i) catabolite-derepressed synthesis of the mRNAs occurs rapidly after a drop in glucose, is a sustained response, and does not require de novo protein synthesis; (ii) heat shock transcription of both invertase mRNAs is, in contrast, a brief and transient response requiring de novo protein synthesis; and (iii) alpha-mating hormone treatment (G1 phase arrest and release) results in regular and coordinated synthesis of both mRNAs midway between rounds of histone mRNA synthesis. We propose that invertase mRNA regulation involves constitutively synthesized transcriptional factors (observed during catabolite derepression) and transient factors (observed during heat shock and possibly during synchronous growth). Moreover, the mRNA levels for secreted and cytoplasmic invertase can be independently regulated.
We analyzed the upstream region of the GDH2 gene, which encodes the NAD-linked glutamate dehydrogenase in Saccharomyces cerevisiae, for elements important for the regulation of the gene by the nitrogen source. The levels of this enzyme are high in cells grown with glutamate as the sole source of nitrogen and low in cells grown with glutamine or ammonium. We found that this regulation occurs at the level of transcription and that a total of six sites are required to cause a CYC1-lacZ fusion to the GDH2 gene to be regulated in the same manner as the NAD-linked glutamate dehydrogenase. Two sites behaved as upstream activation sites (UASs). The remaining four sites were found to block the effects of the two UASs in such a way that the GDH2-CYC1-lacZ fusion was not expressed unless the cells containing it were grown under conditions favorable for the activity of both UASs. This complex regulatory system appears to account for the fact that GDH2 expression is exquisitely sensitive to glutamine, whereas the expression of GLN1, coding for glutamine synthetase, is not nearly as sensitive.
The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.
TSF3 encodes one of six (TSF1 to TSF6) recently identified global negative regulators of transcription in Saccharomyces cerevisiae. Mutant tsf3 strains exhibit defects in transcriptional silencing of the GAL1 promoter, allow expression from upstream activation sequence-less promoters, and exhibit pleiotropic defects in cell growth and development. Here we show that TSF3 is involved in transcriptional silencing mediated by the alpha 2 repressor and demonstrate that specific systems of transcriptional silencing may depend on the more global role of TSF3. Cloning and sequencing of TSF3 allowed us to predict a 974-amino-acid gene product identical to SIN4, a negative regulator of transcription of the HO (homothallism) mating type switching endonuclease. TSF3 disruptions are not lethal but result in phenotypes similar to those of the originally isolated alleles. Our results, together with those of Y. W. Jiang and D. J. Stillman (Mol. Cell. Biol. 12:4503-4514, 1992), suggest that TSF3 (SIN4) affects the function of the basal transcription apparatus, and this effect in turn alters the manner in which the latter responds to upstream regulatory proteins.
The Ty transposable elements of Saccharomyces cerevisiae consist of a single large transcription unit whose expression is controlled by a combination of upstream and downstream regulatory sequences. Errede (B. Errede, Mol. Cell. Biol. 13:57-62, 1993) has shown that among the downstream control sequences is a binding site for the transcription factor, MCM1. A small restriction fragment containing the Ty1 MCM1-binding site exhibits very weak activation of heterologous gene expression. The absence of SPT13 (GAL11) causes a dramatic increase in activity directed by these sequences. This effect is mediated through the MCM1-binding site itself. MCM1 mRNA and protein levels, as well as its affinity for its binding site, are unchanged in the absence of SPT13. Our results suggest that SPT13 has a role in the negative control of MCM1 activity that is likely to be posttranslational. A role for SPT13 in the negative regulation of the activity of the Ty1 MCM1-binding site is consistent with our previous proposal that spt13-mediated suppression of Ty insertion mutations could be attributed to the loss of negative regulation of genes adjacent to Ty elements.
The MSN2 gene was selected as a multicopy suppressor in a temperature-sensitive SNF1 protein kinase mutant of Saccharomyces cerevisiae. MSN2 encodes a Cys2His2 zinc finger protein related to the yeast MIG1 repressor and to mammalian early growth response and Wilms' tumor zinc finger proteins. Deletion of MSN2 caused no phenotype. A second similar zinc finger gene, MSN4, was isolated, and deletion of both genes caused phenotypic defects related to carbon utilization. Overexpression of the zinc finger regions was deleterious to growth. LexA-MSN2 and LexA-MSN4 fusion proteins functioned as strong transcriptional activators when bound to DNA. Functional roles of this zinc finger protein family are discussed.
The cmd1-1 mutation of calmodulin causes temperature-sensitive growth in Saccharomyces cerevisiae. We have isolated a dosage-dependent suppressor of cmd1-1, designated HCM1. Twentyfold overexpression of HCM1 permits strains carrying cmd1-1 to grow at temperatures up to and including 34 degrees C but does not suppress the lethality of either cmd1-1 at higher temperatures or the deletion of CMD1. Thus, overexpression of HCM1 does not bypass the requirement for calmodulin but enhances the ability of the mutant calmodulin to function. HCM1 is not essential for growth, but deletion of HCM1 exacerbates the phenotype of a strain carrying cmd1-1. HCM1 is located on chromosome III, which was recently sequenced. Our results correct errors in the published DNA sequence. The putative polypeptide encoded by HCM1 is 564 amino acids long and has a predicted molecular weight of 63,622. Antisera prepared against Hcm1p detect a protein that is overproduced in yeast strains overexpressing HCM1 and has an apparent molecular mass of 65 kDa. Eighty-six amino acid residues in the N terminus of Hcm1p show 50% identity with a DNA-binding region of the fork head family of DNA-binding proteins. When fused to the DNA-binding domain of Gal4p, residues 139 to 511 of Hcm1p can act as a strong activator of transcription. However, overexpression of HCM1 does not affect the expression of calmodulin. Furthermore, Hcm1p does not bind to calmodulin in a gel overlay assay. Thus, overexpression of HCM1 enhances calmodulin function by an apparently indirect mechanism.
The temperature-sensitive mutation prp20-1 of Saccharomyces cerevisiae exhibits a pleiotropic phenotype associated with a general failure to maintain a proper organization of the nucleus. Its mammalian homolog, RCC1, is not only reported to be involved in the negative control of chromosome condensation but is also believed to assist in the coupling of DNA replication to the entry into mitosis. Recent studies on Xenopus RCC1 have strongly suggested a further role for this protein in the formation or maintenance of the DNA replication machinery. To elucidate the nature of the various components required for this PRP20 control pathway in S. cerevisiae, we undertook a search for multicopy suppressors of a prp20 thermosensitive mutant. Two genes, GSP1 and GSP2, were identified that encode almost identical polypeptides of 219 and 220 amino acids. Sequence analyses of these proteins show them to contain the ras consensus domains involved in GTP binding and metabolism. The levels of the GSP1 transcript are about 10-fold those of GSP2. As for S. cerevisiae RAS2, GSP2 expression exhibits carbon source dependency, while GSP1 expression does not. GSP1 is an essential gene, and GSP2 is not required for cell viability. We show that GSP1p is nuclear, that it can bind GTP in an in vitro assay, and finally, that a mutation in GSP1p which activates small ras-like proteins by increasing the stability of the GTP-bound form causes a dominant lethal phenotype. We believe that these two gene products may serve in regulating the activities of the multicomponent PRP20 complex.
During simian virus 40 lytic infection there is a shift in initiation sites used to transcribe the early region, which encodes large T and small t antigens. Early in infection, transcription is initiated almost exclusively from sites that are downstream of the origin of DNA replication, whereas transcripts produced later are initiated mainly from sites on the upstream side. We have used mutant virus and specially constructed plasmid DNAs to investigate the factors regulating this transcriptional shift. In our studies simian virus 40 large T antigen appears to mediate the shift in transcription in two ways: first, T antigen represses transcription at the downstream sites late in infection by binding to the region where these RNAs are initiated; second, T antigen promotes transcription from sites on the upstream side by its ability to initiate replication or amplification, or both, of the template DNA. In addition, transcription from the downstream sites is heavily dependent on enhancer sequences located in the 72-base-pair repeat region, whereas transcription from the upstream sites late in infection does not require enhancer sequences. Thus, different overlapping promoters regulate simian virus 40 early-region expression in a manner that apparently coordinates the production of large T antigen with the increase in viral DNA.
We have cloned and sequenced the SIN4 gene and determined that SIN4 is identical to TSF3, identified as a negative regulator of GAL1 gene transcription (S. Chen, R.W. West, Jr., S.L. Johnson, H. Gans, and J. Ma, submitted for publication). Yeast strains bearing a sin4 delta null mutation have been constructed and are temperature sensitive for growth and display defects in both negative and positive regulation of transcription. Transcription of the CTS1 gene is reduced in sin4 delta mutants, suggesting that Sin4 functions as a positive transcriptional regulator. Additionally, a Sin4-LexA fusion protein activates transcription from test promoters containing LexA binding sites. The sin4 delta mutant also shows phenotypes common to histone and spt mutants, including suppression of delta insertion mutations in the HIS4 and LYS2 promoters, expression of promoters lacking upstream activation sequence elements, and decreased superhelical density of circular DNA molecules. These results suggest that the sin4 delta mutation may alter the structure of chromatin, and these changes in chromatin structure may affect transcriptional regulation.
We have used degenerate oligonucleotide probes based on sequences conserved among known protein tyrosine phosphatases (PTPases) to identify two Schizosaccharomyces pombe genes encoding PTPases. We previously described the cloning of pyp1+ (S. Ottilie, J. Chernoff, G. Hannig, C. S. Hoffman, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 88:3455-3459, 1991), and here we describe a second gene, called pyp2+. The C terminus of each protein contains sequences conserved in the apparent catalytic domains of all known PTPases. Disruption of pyp2+ results in viable cells, as was the case for pyp1+, whereas disruption of pyp2+ and pyp1+ results in synthetic lethality. Overexpression of either pyp1+ or pyp2+ in wild-type strains leads to a delay in mitosis but is suppressed by a wee1-50 mutation at 35 degrees C or a cdc2-1w mutation. A pyp1 disruption suppresses the temperature-sensitive lethality of a cdc25-22 mutation. Our data suggest that pyp1+ and pyp2+ act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway.
The conserved positions of the eukaryotic cytoplasmic initiator tRNA have been suggested to be important for the initiation of protein synthesis. However, the role of these positions is not known. We describe in this report a functional analysis of the yeast initiator methionine tRNA (tRNA(iMet)), using a novel in vivo assay system which is not dependent on suppressor tRNAs. Strains of Saccharomyces cerevisiae with null alleles of the four initiator methionine tRNA (IMT) genes were constructed. Consequently, growth of these strains was dependent on tRNA(iMet) encoded from a plasmid-derived gene. We used these strains to investigate the significance of the conserved nucleosides of yeast tRNA(iMet) in vivo. Nucleotide substitutions corresponding to the nucleosides of the yeast elongator methionine tRNA (tRNA(MMet)) have been made at all conserved positions to identify the positions that are important for tRNA(iMet) to function in the initiation process. Surprisingly, nucleoside changes in base pairs 3-70, 12-23, 31-39, and 29-41, as well as expanding loop I by inserting an A at position 17 (A17) had no effect on the tester strain. Nucleotide substitutions in positions 54 and 60 to cytidines and guanosines (C54, G54, C60, and G60) did not prevent cell growth. In contrast, the double mutation U/rT54C60 blocked cell growth, and changing the A-U base pair 1-72 to a G-C base pair was deleterious to the cell, although these tRNAs were synthesized and accepted methionine in vitro. From our data, we suggest that an A-U base pair in position 1-72 is important for tRNA(iMet) function, that the hypothetical requirement for adenosines at positions 54 and 60 is invalid, and that a U/rT at position 54 is an antideterminant distinguishing an elongator from an initiator tRNA in the initiation of translation.
Agrobacterium tumefaciens is the causative agent of crown gall disease and is widely used as a vector to create transgenic plants. Under laboratory conditions the yeast Saccharomyces cerevisiae and other yeasts and fungi can also be transformed, and Agrobacterium‐mediated transformation (AMT) is now considered the method of choice for genetic transformation of many fungi. Unlike plants, in S. cerevisiae T‐DNA is integrated preferentially by homologous recombination and integration by non‐homologous recombination is very inefficient. Here we report that upon deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, the efficiency of AMT significantly increased regardless of whether integration of T‐DNA was mediated by homologous or non‐homologous recombination. This correlates with an increase in double‐strand DNA breaks, the putative entry sites for T‐DNA, in the genome of the ada2Δ deletion mutant, as visualized by the number of Rad52‐GFP foci. Our observations may be useful to enhance the transformation of species that are difficult to transform.
Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light-control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in Saccharomyces cerevisiae to perturb both intercellular and interspecies interactions.
Importance
Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition and resilience of microbial communities. In order to better understand the role of these interactions in governing community development it is critical to be able to alter them in a controlled manner. Optogenetically-controlled interactions offer advantages over static perturbations or chemically-controlled interactions as light can be manipulated in space and time and doesn’t require the addition of nutrients or antibiotics. Here we report a system for rapidly achieving light-control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing Saccharomyces cerevisiae and serves as a proof-of-principle for implementing this approach in other consortia.
The SUC2 gene of Saccharomyces cerevisiae encodes two differently regulated mRNAs (1.8 and 1.9 kilobases) that differ at their 5' ends. The larger RNA encodes a secreted, glycosylated form of invertase and the smaller RNA encodes an intracellular, nonglycosylated form. We have determined the nucleotide sequence of the amino-terminal coding region of the SUC2 gene and its upstream flanking region and have mapped the 5' ends of the SUC2 mRNAs relative to the DNA sequence. The 1.9-kilobase RNA contains a signal peptide coding sequence and presumably encodes a precursor to secreted invertase. The 1.8-kilobase RNA does not include the complete coding sequence for the signal peptide. The nucleotide sequence data prove that SUC2 is a structural gene for invertase, and translation of the coding information provides the complete amino acid sequence of an S. cerevisiae signal peptide.
The rate of transcription of murine mammary tumor virus (MTV) sequences in MTV-infected rat hepatoma tissue culture cells is strongly affected by both glucocorticoid hormones and the chromosomal position of provirus integration. We have characterized MTV RNAs produced in J2.17 and M1.54, independent isolates containing, respectively, 1 and 10 proviruses integrated at distinct chromosomal loci. M1.54, but not J2.17, synthesized MTV RNA in the absence of glucocorticoids; the rate of hormone-stimulated viral gene transcription in M1.54 was 50- to 100-fold higher than in J2.17. In each case in which MTV genes were expressed (J2.17 induced, M1.54 basal and induced), the viral RNAs produced were indistinguishable. RNA blotting revealed accumulation of two transcripts, 7.8 and 3.8 kilobases; the latter was likely produced from the former by RNA splicing. Sites used for transcription initiation, polyadenylation, and splicing have been identified from the sizes of end-labeled hybridization probes protected from digestion with mung bean nuclease; the unique initiation and polyadenylation sites were both encoded within the MTV long-terminal-repeat sequence. The efficiencies of splicing and of utilization of the polyadenylation signal did not appear to vary as functions of chromosomal position or hormonal stimulation. Differences in rates of viral gene transcription were reflected in the differential accumulation of the 5'-terminal 136 nucleotides of MTV RNA. Thus, glucocorticoids and chromosomal position appeared to affect solely the efficiency of utilization of the MTV promoter, leaving unchanged the sites of initiation, splicing, and polyadenylation, as well as the efficiencies of the latter two processes.
The chromosomal region containing a structural gene for the mating pheromone precursor prepro-alpha-factor was examined in a variety of Saccharomyces yeasts by using a cloned putative prepro-alpha-factor gene of Saccharomyces cerevisiae as the probe. Analysis by restriction endonuclease digestion and Southern blot hybridization indicated that the physical arrangement of this region is highly conserved in all the Saccharomyces species analyzed, but displays length polymorphisms of limited size (50 to 60 base pairs). The observed polymorphisms were shown to be due solely to differences in the number of tandemly arranged spacer peptide/pheromone units within the coding sequence of these genes. Analysis of polyadenylated RNA indicated that these genes specified RNA transcripts and that these RNA molecules could be translated in vitro into prepro-alpha-factor polypeptides immunoprecipitable with anti-alpha-factor antibodies. The sizes of both the mRNAs and the proteins synthesized from them reflected exactly the differences observed in the lengths of the genes. These findings demonstrate conclusively that the putative prepro-alpha-factor DNA cloned from S. cerevisiae, as well as the sequences detected in the other Saccharomyces species, are indeed expressed and functional genes, and suggest that proper proteolytic processing of prepro-alpha-factor is unaffected by the number of pheromone repeats encoded within this precursor protein.
The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains.
GCN2 is a protein kinase that stimulates translation of GCN4 mRNA in amino acid-starved cells by phosphorylating the alpha subunit of translation initiation factor 2 (eIL-2). We isolated multicopy plasmids that overcome the defective derepression of GCN4 and its target genes caused by the leaky mutation gcn2-507. One class of plasmids contained tRNA(His) genes and conferred efficient suppression only when cells were starved for histidine; these plasmids suppressed a gcn2 deletion much less efficiently than they suppressed gcn2-507. This finding indicates that the reduction in GCN4 expression caused by gcn2-507 can be overcome by elevating tRNA(His) expression under conditions in which the excess tRNA cannot be fully aminoacylated. The second class of suppressor plasmids all carried the same gene encoding a mutant form of tRNA(Val) (AAC) with an A-to-G transition at the 3' encoded nucleotide, a mutation shown previously to reduce aminoacylation of tRNA(Val) in vitro. In contrast to the wild-type tRNA(His) genes, the mutant tRNA(Val) gene efficiently suppressed a gcn2 deletion, and this suppression was independent of the phosphorylation site on eIF-2 alpha (Ser-51). Overexpression of the mutant tRNA(Val) did, however, stimulate GCN4 expression at the translational level. We propose that the multicopy mutant tRNA(Val) construct leads to an accumulation of uncharged tRNA(Val) that derepresses GCN4 translation through a pathway that does not involve GCN2 or eIF-2 alpha phosphorylation. This GCN2-independent pathway was also stimulated to a lesser extent by the multicopy tRNA(His) constructs in histidine-deprived cells. Because the mutant tRNA(Val) exacerbated the slow-growth phenotype associated with eIF-2 alpha hyperphosphorylation by an activated GCN2c kinase, we suggest that the GCN2-independent derepression mechanism involves down-regulation of eIF-2 activity.
The yeast GAL80 gene, encoding a negative regulatory protein of galactose-inducible genes, shows both constitutive and galactose-inducible expression. The inducible transcription is under the control of Gal4p, a common activator for the galactose-inducible genes, which binds to an upstream activation sequence, called UASG, spanning between -105 and -89 in the 5'-flanking region of GAL80. Here we demonstrate that the constitutive transcription started at +1, whereas the inducible transcription occurs from a set of downstream sites at +37, +47, +56, and +67. Both transcriptions were enhanced 10-fold by another UAS, whose 5' boundary is located between -195 and -185. Gal4p stimulated transcription, which depends on the TATA box located at -20, from all the downstream sites. By contrast, the constitutive transcription depended on a small region of less than 16 bp long encompassing the +1 site, which directed transcription even in the absence of both the TATA box and the UASs. When a fragment covering that region was inserted immediately upstream of the open reading frame of HIS3, the resulting gene fusion, if introduced into a his3 yeast strain, supported growth on histidine-lacking medium. We detected by gel retardation assay a protein specifically interacting with this fragment. All the transcriptions observed in the in vivo experiments were faithfully reproduced in a cell-free transcription system. From these results, we suggest that initiation of GAL80 transcription involves two alternative pathways; one is initiator dependent, and the other is Gal4p regulated and TATA dependent.
Clathrin is important but not essential for yeast cell growth and protein secretion. Diploid Saccharomyces cerevisiae cells heterozygous for a clathrin heavy-chain gene (CHC1) disruption give rise to viable, slow-growing, clathrin heavy-chain-deficient meiotic progeny (G. Payne and R. Schekman, Science 230:1009-1014, 1985). The possibility that extragenic suppressors account for growth of clathrin-deficient cells was examined by deletion of CHC1 from haploid cell genomes by single-step gene transplacement and independently by introduction of a centromere plasmid carrying the complete CHC1 gene into diploid cells before eviction of a chromosomal CHC1 locus and subsequent tetrad analysis. Both approaches yielded clathrin-deficient haploid strains. In mutants missing at least 95% of the CHC1 coding domain, transcripts related to CHC1 were not detected. The time course of invertase modification and secretion was measured to assess secretory pathway functions in the viable clathrin-deficient cells. Core-glycosylated invertase was converted to the mature, highly glycosylated form at equivalent rates in mutant and wild-type cells. Export of mature invertase from mutant cells was delayed but not prevented. Abnormal vacuoles, accumulated vesicles, and Golgi body-derived structures were visualized in mutant cells by electron microscopy. We conclude that extragenic suppressors do not account for the viability of clathrin-deficient cells and, furthermore, that many standard laboratory strains can sustain a CHC1 disruption. Clathrin does not appear to mediate protein transfer from the endoplasmic reticulum to the Golgi body but may function at a later stage of the secretory pathway.
The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.
The Saccharomyces cerevisiae SNF2 gene affects the expression of many diversely regulated genes and has been implicated in transcriptional activation. We report here the cloning and characterization of STH1, a gene that is homologous to SNF2. STH1 is essential for mitotic growth and is functionally distinct from SNF2. A bifunctional STH1-beta-galactosidase protein is located in the nucleus. The predicted 155,914-Da STH1 protein is 72% identical to SNF2 over 661 amino acids and 46% identical over another stretch of 66 amino acids. Both STH1 and SNF2 contain a putative nucleoside triphosphate-binding site and sequences resembling the consensus helicase motifs. The large region of homology shared by STH1 and SNF2 is conserved among other eukaryotic proteins, and STH1 and SNF2 appear to define a novel family of proteins related to helicases.
rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.
Mutations in the SSN6 gene suppress the invertase derepression defect caused by a lesion in the SNF1 protein kinase gene. We cloned the SSN6 gene of Saccharomyces cerevisiae and identified its 3.3-kilobase poly(A)-containing RNA. Disruption of the gene caused phenotypes similar to, but more severe than, those caused by missense mutations: high-level constitutivity for invertase, clumpiness, temperature-sensitive growth, alpha-specific mating defects, and failure to homozygous diploids to sporulate. In contrast, the presence of multiple copies of SSN6 interfered with derepression of invertase. An ssn6 mutation was also shown to cause glucose-insensitive expression of a GAL10-lacZ fusion and maltase. The mating defects of MAT alpha ssn6 strains were associated with production of two a-specific products, a-factor and barrier, and reduced levels of alpha-factor; no deficiency of MAT alpha 2 RNA was detected. We showed that ssn6 partially restored invertase expression in a cyr1-2 mutant, although ssn6 was clearly not epistatic to cyr1-2. We also determined the nucleotide sequence of SSN6, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). Possible functions of the SSN6 product are discussed.
LTE1 belongs to the CDC25 family that encodes a guanine nucleotide exchange factor for GTP-binding proteins of the ras family. Previously we have shown that LTE1 is essential for termination of M phase at low temperatures. We have identified TEM1 as a gene that, when present on a multicopy plasmid, suppresses the cold-sensitive phenotype of lte1. Sequence analysis of TEM1 and GTP-binding analysis of the gene product revealed that TEM1 encodes a novel low-molecular-weight GTP-binding protein. The defect of TEM1 was lethal, and the tem1-defective cells were arrested at telophase with high H1-kinase activity under restrictive conditions, indicating that TEM1 is required to exit from M phase. The defect of TEM1 was suppressed by a high dose of CDC15, which encodes a protein kinase homologous to mitogen-activated protein kinase kinase kinases. The genetic interaction among LTE1, TEM1, and CDC15 indicates that they cooperatively play an essential role for termination of M phase.
Induction of the lactose-galactose regulon is strongly repressed by glucose in some but not all strains of Kluyveromyces lactis. We show here that in strongly repressed strains, two to three times less Kl-GAL4 mRNA is synthesized and that expression of structural genes in the regulon such as LAC4, the structural gene for beta-galactosidase, is down regulated 40-fold or more. Comparative analysis of strains having a strong or weak repression phenotype revealed a two-base difference in the promoter of the Kl-GAL4 (also called LAC9) positive regulatory gene. This two-base difference is responsible for the strong versus the weak repression phenotype. The two base changes are symmetrically located in a DNA sequence having partial twofold rotational symmetry (14 of 21 bases). We hypothesize that this region functions as a sensitive regulatory switch, an upstream repressor sequence (URS). According to our model, the presence of glucose in the culture medium signals, by an unidentified pathway, a repressor protein to bind the URS. Binding reduces transcription of the Kl-GAL4 gene so that the concentration of the Kl-GAL4 protein falls below the level needed for induction of LAC4 and other genes in the regulon. For strains showing weak glucose repression, we hypothesize that the two base changes in the URS reduce repressor binding so that the regulon is not repressed. Our results illustrate an important principle of genetic regulation: a small (2- to 3-fold) change in the concentration of a regulatory protein can produce a large (40-fold or greater) change in expression of structural genes. This mechanism of signal amplification could play a role in many biological phenomena that require regulated transcription.
The COT1 gene of Saccharomyces cerevisiae has been isolated as a dosage-dependent suppressor of cobalt toxicity. Overexpression of the COT1 gene confers increased tolerance to cobalt and rhodium ions but not other divalent cations. Strains containing null alleles of COT1 are viable yet more sensitive to cobalt than are wild-type strains. Transcription of COT1 responds minimally to the extracellular cobalt concentration. Addition of cobalt ions to growth media results in a twofold increase in COT1 mRNA abundance. The gene encodes a 48-kDa protein which is found in mitochondrial membrane fractions of cells. The protein contains six possible membrane-spanning domains and several potential metal-binding amino acid residues. The COT1 protein shares 60% identity with the ZRC1 gene product, which confers resistance to zinc and cadmium ions. Cobalt transport studies indicate that the COT1 product is involved in the uptake of cobalt ions yet is not solely responsible for it. The increased tolerance of strains containing multiple copies of the COT1 gene is probably due to increased compartmentalization or sequestration of the ion within mitochondria.
RHO3 and RHO4 are members of the ras superfamily genes of the yeast Saccharomyces cerevisiae and are related functionally to each other. Experiments using a conditionally expressed allele of RHO4 revealed that depletion of both the RHO3 and RHO4 gene products resulted in lysis of cells with a small bud, which could be prevented by the presence of osmotic stabilizing agents in the medium. rho3 rho4 cells incubated in medium containing an osmotic stabilizing agent were rounded and enlarged and displayed delocalized deposition of chitin and delocalization of actin patches, indicating that these cells lost cell polarity. Nine genes whose overexpression could suppress the defect of the RHO3 function were isolated (SRO genes). Two of them were identical with CDC42 and BEM1, bud site assembly genes involved in the process of bud emergence. A high dose of CDC42 complemented the rho3 defect, whereas overexpression of RHO3 had an inhibitory effect on the growth of mutants defective in the CDC24-CDC42 pathway. These results, along with comparison of cell morphology between rho3 rho4 cells and cdc24 (or cdc42) mutant cells kept under the restrictive conditions, strongly suggest that the functions of RHO3 and RHO4 are required after initiation of bud formation to maintain cell polarity during maturation of daughter cells.
The alpha 2 protein of the yeast Saccharomyces cerevisiae normally represses a set of cell-type-specific genes (the a-specific genes) that are transcribed by RNA polymerase II. In this study, we determined whether alpha 2 can affect transcription by other RNA polymerases. We find that alpha 2 can repress transcription by RNA polymerase I but not by RNA polymerase III. Additional experiments indicate that alpha 2 represses RNA polymerase I transcription through the same pathway that it uses to repress RNA polymerase II transcription. These results implicate conserved components of the transcription machinery as mediators of alpha 2 repression and exclude several alternate models.
We have used a modification of the Berk-Sharp technique to determine that the 5′ termini of the mouse 15 S β-globin precursor
and the mature mRNA have identical map coordinates. The modification involves the use of 5′ (or 3′) terminally labeled probes;
it allows the detection of the precursor in the presence of excess nature mRNA.
We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.
Because 50% of the mass of the external invertase of Saccharomyces cerevisiae consists of carbohydrate, it has been extremely difficult to obtain an accurate molecular weight of this enzyme by centrifugal or electrophoretic techniques. However, on removing almost all of the oligosaccharide chains of this enzyme with the endo-beta-N-acetyl-glucosaminidase H from Streptomyces plicatus, it has been possible to show that carbohydrate-free invertase is composed of two 60,000-dalton subunits. Terminal sequence analysis with carboxypeptidases A, B, and Y provided strong evidence that the subunits are identical.
Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6. 19, 6.06
and 5.97 kilobases as referenced to sequenced ØX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA.
Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid
techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share
basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique Hpal site and by having small overlapping deletions in the same region. The Hpal site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of
the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated
on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.
Thirty-seven independently cloned segments of Drosophila melanogaster DNA (Dm segments) were individually tested for their ability to promote the synthesis of new polypeptides in Escherichia coli K-12. The cloning vector was the pSC101 plasmid and the test system consisted of E. coli K-12 minicells that contained the hybrid pDm plasmids. Each of four pDm plasmids produced a new polypeptide, and one, pDm107, was selected for detailed mapping of the sequences required for the translation of its 38,000-dalton polypeptide, the Dm107 protein. Mapping was accomplished by constructing (i) deletion derivatives of pDm107 and (ii) new plasmids consisting of fragments of the Dm107 segment inserted into other vectors, and then testing these hybrids for their ability to promote the synthesis of the Dm107 protein, or truncated versions of this protein, in minicells. The 1000 base pairs of sequences that are translated to yield the Dm107 protein were thereby mapped at the center of the 18,000-base pair Dm107 segment, which consists of nonrepetitive sequences located at the base of the right arm of chromosome 2. The four polypeptides produced by the four pDm plasmids require sequences of 4000 base pairs for their translation, and the total amount of DNA in the 37 cloned Dm segments that were tested is approximately 400,000 base pairs. Because no new polypeptides were detected with the remaining 33 pDm plasmids, the fraction of D. melanogaster sequences that can be efficiently translated in E. coli K-12 is estimated to be 1 x 10(-2).
Most of the studies on yeast invertase have been concerned only with the external enzyme (a mannan protein which is localized in the cell wall), but recently it has been reported that the invertase inside the cytoplasmic membrane is a smaller form. This paper presents a comparative study of the distribution of invertase isozymes in two stains of yeast and, after the selection of the best conditions and source, the purification of the internal or small enzyme. The major purification is obtained by chromatography on diethylaminoethyl Sephadex, although ammonium sulfate precipitation and gel filtration in Sephadex G-200 are also used to separate the large and small enzymes. The purified internal invertase is homogeneous on the basis of chromatographic criteria and its behavior in the analytical ultracentrifuge. The molecular weight of the enzyme is approximately 135,000, and the specific activity is 2,900 units per mg of protein.
Utilization of sucrose as a source of carbon and energy in yeast (Saccharomyces) is controlled by the classical SUC genes, which confer the ability to produce the sucrose-degrading enzyme invertase (Mortimer and Hawthorne 1969). Mutants of S. cerevisiae strain S288C (SUC2 +) unable to grow anaerobically on sucrose, but still able to use glucose, were isolated. Two major complementation groups were identified: twenty-four recessive mutations at the SUC2 locus (suc2 -); and five recessive mutations defining a new locus, SNF1 (for sucrose nonfermenting), essential for sucrose utilization. Two minor complementation groups, each comprising a single member with a leaky sucrose-nonfermenting phenotype, were also identified. The suc2 mutations isolated include four suppressible amber mutations and five mutations apparently exhibiting intragenic complementation; complementation analysis and mitotic mapping studies indicated that all of the suc2 mutations are alleles of a single gene. These results suggest that SUC2 encodes a protein, probably a dimer or multimer. No invertase activity was detected in suc2 mutants.—The SNF1 locus is not tightly linked to SUC2. The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2 + and SUC7 + strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. Although snf1 mutants thus display a petite phenotype, classic petite mutations do not interfere with utilization of sucrose, galactose or maltose. A common feature of all the carbon utilization systems affected by SNF1 is that all are regulated by glucose repression. The snf1 mutants were found to produce the constitutive nonglycosylated form of invertase, but failed to produce the glucose-repressible, glycosylated, secreted invertase. This failure cannot be attributed to a general defect in production of glycosylated and secreted proteins because synthesis of acid phosphatase, a glycosylated secreted protein not subject to glucose repression, was not affected by snf1 mutations. These findings suggest that the SNF1 locus is involved in the regulation of gene expression by glucose repression.
During differentiation, B lymphocytes undergo a shift from expression of membrane-bound IgM to IgM secretion. The μ chains of membrane and secreted IgM, μm and μs, respectively, differ in the amino acid sequence of their carboxy terminal regions. In this paper, we demonstrate that μm and μs heavy chains are encoded by separate mRNAs of 2.7 and 2.4 kb, respectively. Restriction mapping and sequence analysis of μ cDNA clones from a myeloma tumor that produces both types of μ chain indicate that the μm and μs mRNAs are identical throughout the coding region up to the 3′ end of the fourth constant region (Cμ4) domain, but differ in their C terminal coding and 3′ untranslated segments. From the nucleotide sequence of the μm cDNA clone, we predict the amino acid sequence of the 41-residue μm C terminal segment or “M” (membrane) segment. This sequence has characteristics consistent with its being a transmembrane peptide. Thus the μs chain has a 20-residue hydrophilic C terminal segment after the Cμ4 domain, and the μm chain has a 41-residue C terminal segment containing a hydrophobic sequence. We propose that comparable C terminal segments also will be found in other membrane-bound immunoglobulin heavy chains.
As shown in the accompanying paper, μ chains of the membrane-bound ( μ m ) and secreted ( μ s ) forms of IgM are encoded by two species of mRNA. Cloned cDNAs produced from the two μ mRNAs of M104E mouse myeloma tumors differ only at their 3′ ends, which encode either the μ m or μ s C terminus. In this paper, we show that both μ m and μ s mRNAs are produced from transcripts of a single μ gene. The last 187 nucleotides of μ s mRNA are derived from DNA contiguous with the 3′ end of the sequence encoding the C μ 4 domain. The μ m cDNA clone does not include these 187 nucleotides, but instead contains 392 nucleotides derived from two exons located 1850 bp 3′ to the C μ 4 sequence. Comparison of genomic and cDNA sequences show that in μ m mRNA, an RNA splice of 1850 nucleotides joins a site in the coding sequence at the end of C μ 4 with a site at the beginning of the first membrane-specific exon. A second RNA splice of 118 nucleotides joins sequences transcribed from the first and second membrane-specific exons. The differences observed between μ m and μ s cDNAs suggest that developmental control of the site at which poly(A) is added to transcripts of the μ gene determines the relative levels of μ m or μ s chain synthesis. We discuss possible models for the control of μ gene transcripts and the significance of this form of developmentally regulated RNA processing for the evolution of eucaryotic "split genes."
Messenger RNA in animal cells appears to be generated by cleavage of high molecular weight precursors in the nucleus. These mRNA precursors are contained in the metabolically active pool of RNA molecules called heterogeneous nuclear RNA (HnRNA). Polysomal mRNA as well as a substantial part of the HnRNA have been shown to contain 180–200 nucleotide-long segments of poly(A) at the 3' terminus of the polynucleotide chains. In addition to this there is a nonpolysomal pool of ribonuclear protein particles in the cytoplasm that also contains polyadenylated RNA. At present this pool is thought to contain mRNA en route to translation.
Nine sucrose nonfermenting mutants have been isolated from yeast strain EK-6B, carrying the tightly linked SUC3 and MAL3 genes. These mutants are allelic to the SUC3 gene recessive in nature and none of them has detectable levels of either internal or external invertase. A single point mutation leading to the loss of both invertases suggests that either SUC3 is a control gene or codes for a polypeptide which is shared by both invertases.
The arrangement of the coding sequences for the 5 S, 5.8 S, 18 S and 25 S ribosomal RNA from Saccharomyces cerevisiae was analyzed in λ-yeast hybrids containing repeating units of the ribosomal DNA. After mapping of restriction sites, the positions of the coding sequences were determined by hybridization of purified rRNAs to restriction fragments, by R-loop analysis in the electron microscope, and by electrophoresis of S1 nuclease-treated rRNA/rDNA hybrids in alkaline agarose gels. The R-loop method was improved with respect to the length calibration of RNA/DNA duplexes and to the spreading conditions resulting in fully extended 18 S and 25 S rRNA R-loops. The qualitative results are: (1) the 5 S rRNA genes, unlike those in higher eukaryotes, alternate with the genes of the precursor for the 5.8 S, 18 S and 25 S rRNA; (2) the coding sequence for 5.8 S rRNA maps, as in higher eukaryotes, between the 18 S and 25 S rRNA coding sequences. The quantitative results are: (1) the tandemly repeating rDNA units have a constant length of 9060 ± 100 nucleotide pairs with one SstI, two HindIII and, dependent on the strain, six or seven EcoRI sites; (2) the 18 S and 25 S rRNA coding regions consist of 1710 ± 80 and 3360 ± 80 nucleotide pairs, respectively; (3) an 18 S rRNA coding region is separated by a 780 ± 70 nucleotide pairs transcribed spacer from a 25 S rRNA coding region. This is then followed by a 3210 ± 100 nucleotide pairs mainly non-transcribed spacer which contains a 5 S rRNA gene.
A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a matingtype-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.
We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 50 degrees. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double- and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel.
This chapter discusses the process of detection of specific ribonucleic acids (RNAs) or specific fragments of deoxyribonucleic acid (DNA) by fractionation in gels and transfer to diazobenzyloxymethyl (DBM) paper. Small, double-stranded fragments of DNA (30–1800 base pairs) are separated with high resolution on composite gels of agarose and polyacrylamide cross-linked with N,N'-diallyltartardiamide instead of N,N′-methylenebisacrylamide. Following electrophoresis, the cleavage of the crosslinks with periodic acid facilitates the transfer of the denatured fragments to DBM paper. Detection is accomplished with labeled probes. Large fragments of DNA can also be transferred from agarose gels to DBM paper, following partial depurination with dilute acid and strand cleavage with NaOH. The efficiency of transfer is very high and independent of the size of a fragment as covalent linkage allows convenient multiple reuse of the transfers and also allows washings to be done repeatedly under stringent conditions, thereby helping to achieve very low backgrounds. Although many kinds of filter paper can be used, Whatman 540 paper was used in the study described in the chapter because of its excellent mechanical strength and resistance to chemicals. Schleicher and Schuell 589 WH paper also gives good results.
A method for purifying sequences adjacent to satellite DNA in the heterochromatin of D. melanogaster is described. A cloned DNA segment containing part of a copia gene adjacent to 1.688 g/cm3 satellite DNA has been isolated. The copia genes compose a repeated gene family which codes for abundant cytoplasmic poly(a)-containing RNA (Young and Hogness, 1977; Finnegan et al., 1978). We have identified two major poly (A)-containing RNA species [5.2 and 2.1 kilobases (kb)] produced by the copia gene family. The cloned segment contains copia sequences homologous to the 5' end of RNA within 0.65 kb of the 1.688 satellite DNA sequences. Seven different cloned copia genes from elsewhere in the genome have also been isolated, and a 5.2 kb region present in five of the clones was identified as copia by heteroduplex analysis. In addition, three ususual copies of copia were found: a "partial" copy of the gene (3.7 kb) which has one endpoint in common with the 5.2 kb unit; a copia gene flanked on one side by a 1.6 kb sequence and on the other by the same 1.6 kb sequence in the inverted orientation; and a copia gene flanked only on one side by the same sequence.
DNA from simian virus 40 (SV40) was prepared for local mutagenesis by nicking the molecule at a specific site with a restriction endonuclease that recognizes one site in SV40 DNA and then extending the nick enzymatically to expose a short, single-stranded segment of DNA. The "gapped" DNA was treated with a single-strand-specific mutagen, sodium bisulfite, which converts cytosine to uracil. After mutagenesis, the gap was repaired with DNA polymerase, generating molecules resistant to the restriction enzyme used to make the initial nick. From cells infected with DNA thus modified, SV40 mutants were isolated that had enzyme-resistant genomes. In some cases, precise positions of G.C to A.T transitions could be inferred from the patterns of susceptibility of mutant DNA to other restriction endonucleases whose recognition sequences were altered by the mutagenesis procedure. One of the restriction endonuclease sites mutagenized (Bgl I) maps at the origin of SV40 DNA replication and near sequences corresponding to the 5' ends of viral mRNAs. Many of the resulting Bgl I-resistant mutants yielded small plaques, suggesting partial defectiveness in DNA replication or transcription.
We have examined the pattern of synthesis of the glycoprotein form of invertase and of the smaller carbohydratefree from in synchronous culture to obtain further infromation concerning their biosynthetic relationship. Saccharomyces mutant 1710 was chosen since its invertase production is almost completely derepressed during growth in 0.1 M mannose medium. The large enzyme, unlike the small form, binds to concanavalin A-Sepharose, and on this basis the two types can conveniently be separated for analysis. Large invertase was produced throughout the cell cycle. Synthesis of the small invertase was periodic; the single burst occurred at or close to the budding stage. Tunicamycin, which inhibits the sypthesis of external glycoproteins, halted formation of the large enzyme but not of the small form, and there was no accumulation of invertase activity with the properties of the small enzyme. Hence, it is unlikely that the small form is a precursor of the large one. Despite marked differences in their amino acid compositions, the two enzymes have many similarities. They are probably, in part, the products of the same gene(s), and the differences between them may largely reflect differences in post-translational processing.
In vitro recombination techniques were used to construct a new cloning vehicle, pBR322. This plasmid, derived from pBR313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin E1, and carries resistance genes to the antibiotics ampicillin (Ap) and tetracycline (Tc). The antibiotic-resistant genes on pBR322 are not transposable. The vector pBR322 was constructed in order to have a plasmid with a single PstI site, located in the ampicillin-resistant gene (Apr), in addition to four unique restriction sites, EcoRI, HindIII, BamHI and SalI. Survival of Escherichia coli strain X1776 containing pBR313 and pBR322 as a function of thymine and diaminopimelic acid (DAP) starvation and sensitivity to bile salts was found to be equivalent to the non-plasmid containing strain. Conjugal transfer of these plasmids in bi- and triparental matings were significantly reduced or undetectable relative to the plasmid ColE1.
A stable leu2- yeast strain has been transformed to LEU2+ by using a chimeric ColE1 plasmid carrying the yeast leu2 gene. We have used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced. These studies show that ColE1 DNA together with the yeast sequences can integrate into the yeast chromosomes. This integration may be additive or substitutive. The bacterial plasmid sequences, once integrated, behave as a simple Mendelian element. In addition, we have determined the genetic linkage relationships for each newly introduced LEU2+ allele with the original leu2- allele. These studies show that the transforming squences integrate not only in the leu2 region but also in several other chromosomal locations.
Polyacrylamide gel electrophoresis (without SDS) of invertases from strains each carrying only one of the five known SUC-genes revealed differences in mobility of the internal enzymes. SUC1 invertase moved distinctly slower than the invertases formed in the presence of genes SUC2 to SUC5. Three bands of internal invertase activity were found in diploids carrying both SUC1 (slow invertase) and one of the other SUC-genes (fast invertases). Tetrad analysis of such diploids yielded haploids which showed the same three bands if they carried SUC1 in combination with another SUC gene. A gene dosage effect was observed in relation to invertase activity in haploid strains with only gene SUC1 or only SUC4 on one hand, and both genes on the other hand. A sucrose non-fermenting and invertase negative strain with mutant allele suc3-3 of gene SUC3 (fast invertase) was crossed with SUC1. The heterozygous diploid and the recombinant haploids (SUC1 suc3-3) showed two bands in the region of the internal invertase: a slow SUC1 band and a second band corresponding to the intermediate band of SUC1-SUC3 strains. The intermediate band in SUC1 suc3-3 strains is considered as a hybrid consisting of an active SUC1-monomer and an inactive suc3-mutant monomer. Formation of such hybrid bands was taken as evidence for the structural nature of SUC-genes.
The sequence organization of the 1.688 satellite DNA (density 1.688 g/cm3 in CsCl) has been investigated, and this satellite has been found to differ from the other D. melanogaster satellite DNAs in having a much greater sequence complexity. Purification of 1.688 satellite DNA by successive equilibrium density centrifugations yielded a fraction 77% pure. Segments of satellite DNA were isolated by molecular cloning in the plasmid vector pSC101. One recombinant plasmid contained a segment of 1.688 satellite DNA 5.8 kilobase pairs in size and was stable during propagation in E. coli. Recognition sites for restriction enzymes from Haemophilus aegyptius (Hae III), Haemophilus influenzae f (Hinf) and Arthrobacter luteus (Alu I) were mapped in the satellite DNA of this hybrid plasmid. The spacing of Hae III, Hinf and two Alu I sites at regular intervals of about 365 base pairs is strong evidence that the sequence complexity of this satellite DNA is 365 base pairs. Further evidence comes from the finding that both gradient-purified and cloned 1.688 satellite DNA renature with their Hae III sites in register. The Hae III and Hinf sites in gradient-purified satellite DNA have been shown by Manteuil, Hamer and Thomas (1975) and Shen, Wiesehahn and Hearst (1976) to be distributed at intervals of 365 base pairs and integral multiples thereof. These investigators proposed that some of the sites in an otherwise regular array have been randomly inactivated. Cloned satellite DNA provided a hybridization probe for sensitive studies of the arrangement of these recognition sites in gradient-purified satellite DNA. Some regions of satellite DNA were found to contain many fewer recognition sites than expected from the proposed models. These findings suggest that different regions of 1.688 satellite DNA may exhibit different arrangements of Hae III and Hinf recognition sites.
We describe a technique for transferring electrophoretically separated bands of RNA from an agarose gel to paper strips. The RNA is coupled covalently to diazobenzyloxymethyl groups on the paper. After transfer and appropriate treatment of the paper to destroy remaining diazo groups, specific RNA bands can be detected by hybridization with 32P-labeled DNA probes followed by autoradiography. This procedure allows detection of specific RNA bands with high sensitivity and low background.
When a DNA molecule, enzymatically labelled with 32p at one end, is partially digested with a restriction enzyme labelled tdna fragments are obtained which form an overlapping series of molecules, all with a common labelled terminus. ta restriction map can then be constructed from an analysis of the size distribution of these molecules. This technique has been used for the restriction site mapping of cloned histone DNA (h22) where as many as 35 cleavage sites may be accurately determined in a single experiment.
The DNA of bacteriophage T7 is cut into seven unique fragments by the restriction endonuclease DpnII (or the equivalent MboI), 19 fragments by HpaI, and eight additional fragments by the combination of the two enzymes. The relative location of each fragment in the T7 DNA has been determined by a combination of techniques. If it is assumed that the length of any DNA molecule equals the sum of the lengths of the fragments produced from it by cleavage, and that electrophoretic mobility through agarose gels is a smooth function of the length of the DNA, then the known relationships between fragments provide enough conditions to define accurately the relative molecular weight of each fragment in the set. Absolute molecular weights are based on that of full-length T7 DNA. The fragments provide a convenient set of length standards covering the entire range from about 100 to 40,000 base-pairs (the length of T7 DNA).
A horizontal slab gel system for electrophoresis on agarose gels is described. In this system, gels of very low concentrations do not distort during electrophoresis and accurate relative mobilities of large DNAs are obtained. Excellent resolution can be obtained for DNAs of molecular weights up to at least 26·5×106, a difference of less than 10% being readily resolved even for molecules of this size.
Agarose and polyacrylamide gels can be prepared in alkaline solvents that denature native DNA and completely unfold the single strands. The fragments of T7 DNA have the same relative mobilities whether subjected to electrophoresis as single strands in alkaline gels or as double-stranded DNA in neutral gels, and resolution is comparable in the two states. Thus, electrophoresis in alkaline gels can provide accurate molecular weights for linear, single-stranded DNAs, and should be useful in analyzing DNA for single-strand breaks, depurinations or topological differences such as ring forms.
In both neutral and alkaline gels, the relative mobilities of DNAs shorter than about 1000 base-pairs (or bases) are essentially insensitive to changes in voltage gradient, at least over the range of voltage gradients commonly employed. However, relative mobilities become increasingly sensitive to voltage gradient the larger the DNA, with DNAs longer than about 20,000 base-pairs (or bases) being severely affected. This effect is probably due to the ease with which large DNA molecules can be deformed from their equilibrium conformations, thus permitting them to penetrate channels in the gel that would exclude them in their unperturbed conformations. As a practical matter, this means that low voltage gradients must be used for separations of large DNAs by gel electrophoresis.
Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>108 cts/min per μg) in vitro using deoxynucleoside [α-32P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.
We have developed a simple and sensitive method for detecting, sizing and mapping RNA transcripts from viral or cloned DNAs. This technique has been used to examine the cytoplasmic transcripts produced during the early phase of adenovirus 2 (Ad2) infection of HeLa cells. Unlabeled total cytoplasmic or oligo (dT)-selected cytoplasmic RNA is hybridized to restriction fragments of 32P-labeled viral DNA in 80% formamide under conditions above the Tm of the DNA duplex, but below the Tm of the RNA-DNA hybrid duplex (Casey and Davidson, 1977). DNA complements precisely the length of the hybridized RNA are generated by treating with single-strand-specific S1 endonuclease under conditions which do not introduce strand breaks into hybrid duplex. The sizes of the S1-resistant single-stranded DNAs are then determined by alkaline agarose gel electrophoresis (McDonnell, Simon and Studier, 1977). A restriction fragment which terminates within a region coding for an mRNA yields a band equal in size to the portion of the mRNA transcribed from that restriction fragment. This allows unique mapping of coding regions relative to restriction endonuclease cleavage sites.
This chapter discusses a procedure for isolation of DNA from the yeast Saccharomyces cerevisiae. This procedure is a modification of an earlier method for isolating DNA from bacteria and has general operations: (1) preparation of osmotically fragile spheroplasts by enzymatic digestion of the cell wall, (2) lysis of the spheroplasts and partial proteolysis of the lysate, (3) deproteinization and extraction of lipids from the lysate, and (4) enzymatic digestion of RNA and elimination of polysaccharides by centrifugation or digestion, followed by separation of the DNA from the digestion products by selective isopropanol precipitation. This procedure yields a preparation of total-yeast DNA including all three buoyant density species observed in neutral isopycnic cesium chloride gradients: nuclear, nuclear heavy satellite, and mitochondrial. If spheroplasting of the cells is adequate and lysis is complete, there are no preferential losses of any of the observed DNA species. Preparations free of DNA of mitochondrial density can be obtained from petite yeast strains that lack mitochondrial DNA. The various DNA species obtained from normal strains by this method may be separated and purified using cesium chloride gradients, cesium chloride–ethidium bromide gradients, cesium sulfate–mercury, or cesium sulfate–silver gradients, and hydroxyapatite chromatography.
Nine sucrose nonfermenting mutants have been isolated from yeast strain EK-6B, carrying the tightly linked SUC3 and MAL3 genes. These mutants are allelic to the SUC3 gene recessive in nature and none of them has detectable levels of either internal or external invertase. A single point mutation leading to the loss of both invertases suggests that either SUC3 is a control gene or codes for a polypeptide which is shared by both invertases.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
A rapid and sensitive method for the location of enzymes after disc gel electrophoresis has been developed by a modification of the well-known formazan reaction. By means of the same assay, semiquantitative evaluation of the enzymic activity can also be accomplished, permitting measurement of enzymic activity in crude extracts. As has been illustrated by several examples, the procedures can be modified and adapted for the detection and measurement of numerous enzymes. The method is applicable to all enzymes converting nonreducing substrates into products exhibiting reducing properties. In addition, macromolecules susceptible to periodate oxidation can be selectively stained employing the same procedure.
An electrophoretic method has been developed for the analysis of ribonucleic acids (RNAs) ranging in size from 104 to 108 daltons. The method depends on the use of acrylamide gels strengthened with agarose for analysis of the larger RNAs. The resolving power of the method permitted individual characterization of RNAs in mixtures containing multiple species of RNA, without prior purification of each species; RNA molecules which differed in molecular weight by only a few per cent could be clearly distinguished, and the molecular weight of each estimated. This unusual application of electrophoretic methods for the determination of molecular weight is based on the observation that, for RNAs, smaller molecules migrate more rapidly than larger ones. The mobility and the logarithm of the molecular weight are inversely related and this relationship is approximately linear. The molecular weights estimated by this technique, although numerically dependent on values assigned to known RNA standards, are highly reproducible in gels of various composition, and are at present the best means of identification of species resolved by gel electrophoresis. By this means, liver 18S RNA is identified as a doublet of RNAs of 0.66 and 0.62 × 106 daltons and the analogous 16 S of Escherichia coli as a doublet of 0.58 and 0.54 × 106 daltons, while liver 5S RNA has a molecular weight of 38,000 daltons.
A complementation analysis of host-controlled modification and restriction of DNA by Escherichia coli has been carried out by examining the restriction and modification phenotypes of partial, permanent diploids containing various arrangements of wild type and mutant restriction and modification alleles. Intercistronic complementation was observed between three classes of restriction and modification mutants of E. coli B, indicating that at least three cistrons (the ram cistrons) are involved in the genetic control of the [restriction and modification of DNA. Mutations in one cistron (ramA) result in a loss of restriction activity but not in modification activity (r−m+). Mutations in a second cistron (ramC) result in a loss of restriction and modification activities (r−m−). Mutations in a third cistron result in a loss of modification activity and appear to be lethal unless accompanied by a mutation in the ramA or ramC cistrons. A fourth class of mutations, which are linked to the other ram cistrons and are expressed phenotypically as r−m− mutants, are trans dominant to the wild-type ram alleles. It is not known if this latter class of mutants represents a fourth cistron of the ram locus. Complementation was observed between E. coli K12 and B ramA and ramC mutations and the host specificity of the restored restriction activity was dependent on an intact ramC cistron. However, complementation was not detected between the P1 and K12 or P1 and B ram alleles. A general model for the genetic control of the restriction and modification properties of E. coli strains and their episomes is presented.
Direct measurements of the intracellular level of λ repressor have been made by a DNA-filter assay and a radioimmune assay. Transcription of cI, the structural gene for repressor, appears to initiate at two different promoters, prm and pre. Promoter pre is activated during the establishment of lysogeny by the action of cII and cIII proteins at the DNA site cY. Phage mutated in cII, cIII, or cY do not make a normal burst of repressor after infection and do not efficiently lysogenize the cell. Cro product stops repressor synthesis midway in the infective cycle.
Promoter prm maintains the repressor level in established lysogens. Delection mapping places it very near the right operator (Or). Prm is activated by repressor bound to the right operator. In the absence of cII or cIII protein, repressor synthesis requires active repressor and only proceeds on genomes able to bind repressor at Or.
The alpha-amylase mRNAs which accumulate in two different tissues of the mouse, the salivary gland and the liver, are identical except for their 5' non-translated sequences: the 5' terminal 158 nucleotides of the major liver alpha-amylase mRNA are unrelated to the 5' terminal 47 nucleotides found in its salivary gland counterpart. DNA that specifies the 5'terminal one-quarter of these mRNAs has been isolated through genomic cloning and sequenced. The initial 161 nucleotides of the liver alpha-amylase mRNA are specified by DNA sequences that lie 4.5 kb upstream from those for the common body of the two mRNAs. In contrast, the 5' terminal 50 nucleotides of the salivary gland alpha-amylase mRNA are found 7.5 kb from sequences that the two mRNAs share in the genome. These cloned DNA sequences occur once per haploid genome, indicating that both the salivary gland and liver alpha-amylase mRNAs are transcribed from the same gene (Amy1A). Since no rearrangement of these DNA sequences can be detected among mouse sperm, salivary gland or liver preparations, gross rearrangement does not account for the tissue-specific pattern of expression observed for Amy1A. Rather, these data indicate that the salivary gland and liver alpha-amylase mRNAs are differentially transcribed and/or processed from identical DNA sequences in different tissues.
This chapter discusses the sequencing end-labeled DNA with base-specific chemical cleavages. In the chemical DNA sequencing method, one end-labels the DNA, partially cleaves it at each of the four bases in four reactions, orders the products by size on a slab gel, and then reads the sequence from an autoradiogram by noting which base-specific agent cleaved at each successive nucleotide along the strand. This technique sequences the DNA made in and purified from cells. No enzymatic copying in vitro is required, and either single- or double-stranded DNA can be sequenced. Most chemical schemes that cleave at one or two of the four bases involve three consecutive steps: modification of a base, removal of the modified base from its sugar, and DNA strand scission at that sugar. Base-specific chemical cleavage is only one step in sequencing DNA. The chapter presents techniques for producing discrete DNA fragments, end-labeling DNA, segregating end-labeled fragments, extracting DNA from gels, and the protocols for partially cleaving it at specific bases using the chemical reactions. The chapter also discusses the electrophoresis of the chemical cleavage products on long-distance sequencing gels and a guide for troubleshooting problems in sequencing patterns.
Saccharomyces cerevisiae revertant strain D10-ER1 has been shown to contain thermosensitive forms of the large (glycoprotein) and small (carbohydrate-free) invertases and a very low level of the small enzyme, along with a wild-type level of the large form (T. Mizunaga et al., Mol. Cell. Biol. 1:460-468, 1981). These characteristics cosegregated in crosses of the revertant strain with wild-type sucrose-fermenting (SUC1) or nonfermenting (suc0) strains. In addition, there is tight linkage between sucrose and maltose fermentation in revertant D10-ER1 (characteristic of the SUC1 and MAL1 genes). From this we infer that a single reversion event is responsible for the several changes observed in D10-ER1, and that this mutation maps within or very close to the SUC1 gene present in the ancestor strain 4059-358D. The revertant SUC1 allele in D10-ER1 (termed SUC1-R1) was expressed independently of the wild-type SUC1 gene when both were present in diploid cells. Diploids carrying only the wild-type or the mutant genes synthesized invertases with the characteristics of the parental Suc+ haploids. The possibility that a modifier gene was responsible for the alterations in the invertases of revertant D10-ER1 was ruled out by appropriate crosses. We conclude that SUC1 is a structural gene that codes for both the large and the small forms of invertase and suggest that SUC2 through SUC5 are structural genes as well.
During differentiation, B lymphocytes undergo a shift from expression of membrane-bound IgM to IgM secretion. The mu chains of membrane and secreted IgM, mum and mus, respectively, differ in the amino acid sequence of their carboxy terminal regions. In this paper, we demonstrate that mum and mus heavy chains are encoded by separate mRNAs of 2.7 and 2.4 kb, respectively. Restriction mapping and sequence analysis of mu cDNA clones from a myeloma tumor that produces both types of mu chain indicate that the mum and mus mRNAs are identical throughout the coding region up to the 3' end of the fourth constant region (Cmu 4) domain, but differ in their C terminal coding and 3' untranslated segments. From the nucleotide sequence of the mum cDNA clone, we predict the amino acid sequence of the 41-residue mum C terminal segment or "M" (membrane) segment. This sequence has characteristics consistent with its being a transmembrane peptide. Thus the mus chain has a 20-residue hydrophilic C terminal segment after the Cmu 4 domain, and the mum chain has a 41-residue C terminal segment containing a hydrophobic sequence. We propose that comparable C terminal segments also will be found in other membrane-bound immunoglobulin heavy chains.
The mRNA isolated from B lymphocyte tumor cell lines directs synthesis of two forms of μ heavy chain, one with a molecular weight of 67K and one of 64K. When these cell lines are converted to IgM-secreting cells by fusion with a myeloma cell, the 64K form of μ predominates; thus it is designated μs (μ-secreted). The 67K form correlates with the presence of surface IgM; thus it is designated μm (μ-membrane). Cells that make both forms of μ chain have two mRNAs, one of 2.4 kb that encodes μs and one of 2.7 kb that encodes μm. The difference between the μs and μm mRNAs can be localized to their 3′ ends by hybridizing 32P-cDNA copies of the mRNA to a cloned copy of μs mRNA, treating the mixtures with SI nuclease, and resolving the nuclease-resistant duplexes by electrophoresis. By probing the separated species of RNA with a DNA copy of the 3′ untranslated region of μs mRNA, it was shown that the 3′ ends of the two μ mRNAs do not cross-hybridize. The difference between the two RNAs was mapped to the 3′ edge of the Cμ4 domain. Apparently two separate 3′ terminal sequences for μ mRNA are encoded in the genome, one that specifies an amino acid sequence appropriate for membrane-binding and a second that is involved in secretion. At different stages of immunocyte development, different μ mRNAs predominate: μm during the lymphocyte stages and μs during the secretion stages.
The SUC genes of yeast (Saccharomyces) genetically appear to constitute a family of repeated genes that are dispersed in the yeast genome. Each SUC+ gene confers upon the strains carrying it the ability to produce invertase, a primarily extracellular and glycosylated enzyme that cleaves sucrose to yield fructose and glucose. Thus, strains carrying a SUC+ allele can ferment sucrose. An unusual feature of this dispersed gene family is that different Saccharomyces strains (or species) have SUC+ alleles at different chromosomal loci; to date, six (possibly seven) unlinked SUC loci have been identified. Unlike SUC2, the suc alleles at most of the SUC loci in the strains examined do not contain SUC-gene information. This finding suggests that the presence of active SUC genes at these loci in some strains results from movement of SUC information during the evolution of yeast strains. Such movement could have occurred either by a series of gross chromosomal rearrangements or, perhaps, by the transposition of a specific element containing an active SUC+ gene. The suc alleles would then represent either the complete absence of any special information (i.e., just random sequences into which SUC DNA became inserted) or some kind of specific preferred integration site for the postulated specific element. We cannot distinguish between these possibilities at present.
We have studied regulation of invertase putative structural genes (SUC) in S. cerevisiae and the synthetic relationship between secreted, glycosylated invertase (E.C.3.2.1.26) and the cytoplasmic, nonglycosylated form of the enzyme. Using immunoprecipitation and gel electrophoresis, we have analyzed invertase polypeptides and glycopeptides synthesized in vitro and in vivo. Analysis of size-fractionated mRNA from a SUC2 strain has shown that three mature, catabolite-repressible mRNA species direct the in vitro synthesis of three invertase polypeptides that have differing molecular weights. Two of these polypeptides, P63 and P62 (63 and 62 kd), are larger than the polypeptides of the secreted enzyme and are cotranslationally processed by microsomal membranes in vitro to yield secreted invertase glycopeptides (GP90 and GP87). The smallest polypeptide, P60 (60 kd), which comigrates electrophoretically with cytoplasmic invertase, is not processed. Posttranslationally, a microsomal-membrane detergent extract removes approximately 20 aminoacids from P62 but not from P60. In vitro translations of mRNAs from a genetically confirmed suc3 mutant strain, from the parental SUC3 strain and from derivative meiotic segregants have shown that the three polypeptides (and therefore three mRNA species) are encoded by one gene. Analysis of in vivo radiolabeled invertase from the same SUC3 and suc3 strains has verified that the SUC3 locus contains the structural gene for secreted and cytoplasmic invertase. Through the derepressed synthesis of multiple primary or processed transcripts, the SUC2 and SUC3 genes are regulated to produce multiple invertase polypeptides. The larger two polypeptides appear to be processed and secreted to yield glycosylated invertase, while the smallest remains in the cytoplasm.
The SUC genes (SUC1-SUC7) of Saccharomyces are a family of genes that are dispersed in the yeast genome. A SUC+ allele at any locus confers the ability to produce the enzyme invertase and, thus, to ferment sucrose. Most yeast strains do not carry SUC+ alleles at all possible SUC loci. We have investigated the naturally occurring negative (suc0) alleles present at SUC loci with the aim of distinguishing between two possible models for the structure of suc0 alleles: (1) suc0 alleles correspond to a simple absence of SUC genetic information; (2) suc0 alleles are "silent" SUC genes that either produce a defective product or are not expressed. To facilitate these studies, sucrose-nonfermenting strains were constructed that are congenic to S. cerevisiae strain S288C (SUC2+), but carry at the SUC2 locus the naturally occurring negative allele, suc2(0), of strain FL100 (Lacroute 1968). These strains were used to study the genetic properties of the suc2(0) allele of FL100 and the suc0 alleles (suc1(0), suc3(0), etc.) of S288C. The suc2(0) allele was shown to revert to an active Suc+ state and to provide functional information at three points in the SUC2 gene in recombination experiments; this suc2(0) gene thus appears to be a "silent" gene. Similar tests for silent SUC genes in S288C (corresponding to loci other than SUC2) failed to reveal any additional silent genes.
Utilization of sucrose as a source of carbon and energy in yeast (Saccharomyces) is controlled by the classical SUC genes, which confer the ability to produce the sucrose-degrading enzyme invertase (Mortimer and Hawthorne 1969). Mutants of S. cerevisiae strain S288C (SUC2+) unable to grow anaerobically on sucrose, but still able to use glucose, were isolated. Two major complementation groups were identified: twenty-four recessive mutations at the SUC2 locus (suc2-); and five recessive mutations defining a new locus, SNF1 (for sucrose nonfermenting), essential for sucrose utilization. Two minor complementation groups, each comprising a single member with a leaky sucrose-nonfermenting phenotype, were also identified. The Suc2 mutations isolated include four suppressible amber mutations and five mutations apparently exhibiting intragenic complementation; complementation analysis and mitotic mapping studies indicated that all of the suc2 mutations are alleles of a single gene. These results suggest that SUC2 encodes a protein, probably a dimer or multimer. No invertase activity was detected in suc2 probably a dimer or multimer. No invertase activity was detected in suc2 mutants,--The SNF1 locus is not tightly linked to SUC2. The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2+ and SUC7+ strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. Although snf1 mutants thus display a petite phenotype, classic petite mutations do not interfere with utilization of sucrose, galactose or maltose. A common feature of all the carbon utilization systems affected by SNF1 is that all are regulated by glucose repression. The snf1 mutants were found to produce the constitutive nonglycosylated form of invertase, but failed to produce the glucose-repressible, glycosylated, secreted invertase. This failure cannot be attributed to a general defect in production of glycosylated and secreted proteins because synthesis of acid phosphatase, a glycosylated secreted protein not subject to glucose repression, was not affected by snf1 mutations. These findings suggest that the SNF1 locus is involved in the regulation of gene expression by glucose repression.