Showing papers in "Current Genetics in 1991"

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA.

Abstract: We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.

Isolation, characterization and sequence of a gene conferring resistance to the systemic fungicide carboxin from the maize smut pathogen, Ustilago maydis

Abstract: A gene which confers resistance to the systemic fungicide carboxin (Cbx) has been isolated from the maize pathogen, Ustilago maydis, by transferring a plasmid gene library from a Cbx-resistant mutant strain into a sensitive strain and selecting for expression of the resistance gene. Five plasmids, rescued from transformants which exhibited enhanced resistance to Cbx, were shown to have DNA inserts with common restriction enzyme fragments. All the plasmids transformed a sensitive U. maydis strain to Cbx resistance. The gene (Cbx r ), sub-cloned on a 3.2 kb EcoR1-HindIII fragment, transformed U. maydis to Cbx resistance at frequencies similar to those obtained with the bacterial Hygromycin B resistance (HygB r ) gene. The sequence of the Cbx r gene showed a high degree of homology to succinate dehydrogenase (EC 1.3.99.1) iron-sulphur subunit genes from other organisms.

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans.

Abstract: Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.

Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea

Abstract: We report here on the development of a transient expression system for Chlorella ellipsoidea using a heterologous gene, firefly luciferase. Cells of this unicellular green alga were converted to protoplasts and treated with plasmid pDO432, which bears luciferase under the control of the CaMV 35S promoter. This treatment resulted in detectable luciferase activity in cell extracts. Expression required Cellulysin treatment, active cell metabolism, and the addition of carrier DNA and polyethylene glycol. Linearization of the luciferase plasmid did not significantly alter the activity. A time course of expression showed that luciferase is made rapidly, within about 7 h after addition of DNA, but that the activity disappears over the course of a few days. These experiments represent an important first step in the development of a Chlorella transformation system.

The use of RFLP analysis in classification of the black Aspergilli: reinterpretation of the Aspergillus niger aggregate

Abstract: By studying ribosomal banding patterns in ethidium bromide-stained gels of chromosomal digests we are able to provide a rapid and reliable classification of a number of taxonomically important isolates of the black Aspergilli. This classification is supported by Southern blots using several pectin lyase genes, isolated from A. niger CBS 120.49, as probes. Taxonomy on the basis of RFLP analysis leads to a classification which is completely different from, but more reliable than, the current one which is mainly based on morphological characteristics. The 23 Aspergillus niger isolates investigated could be divided into two distinct groups on the basis of our results. We propose that these groups represent two different species: A. niger and A. tubigensis. This is supported by preliminary results showing failure of heterokaryon formation between typical representatives of both groups.

A 2.5 kb ncoi fragment of ogura radish mitochondrial dna is correlated with cytoplasmic male-sterility in brassica cybrids

Abstract: Spontaneous reversion to fertility was studied in the progeny of a cytoplasmic male-sterile (CMS) Brassica napus cybrid containing recombinant B. napus/Ogura radish mitochondrial genomes. This reversion is concomitant with the disappearance of a 2.5 kb NcoI fragment present in the mitochondrial DNA of Ogura radish, and of CMS cybrids derived from plants carrying Ogura cytoplasm, and absent in the mitochondrial genome of normal Brassicas and fertile cybrids. This specific fragment hybridizes to a 1.4 kb transcript found only in male-sterile plants bearing an Ogura derived cytoplasm.

Expression and sequence comparison of the Aspergillus niger and Aspergillus tubigensis genes encoding polygalacturonase II.

Abstract: The structure and expression of the polygalacturonase-encoding pgaII genes of two recently recognized species, Aspergillus niger and Aspergillus tubigensis, was investigated. While the structure of the pgaII genes is very similar, showing 83% DNA sequence identity and 94% identity at the amino acid level, they have diverged significantly. The NH2-terminal sequence suggests that these PGs are made as pre pro-proteins and the secretory propeptide of the PGII precursors shows sequence homology with some other fungal pro-peptides. The expression of the pgaII genes is strongly regulated by the carbon source and the A. tubigensis gene is expressed and regulated in A. niger transformants. The low similarity of the fungal PGs with those of bacterial and plant origin is discussed in relation to the possible functional role of specific amino acids.

The kalilo linear senescence-inducing plasmid of Neurospora is an invertron and encodes DNA and RNA polymerases.

Abstract: The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by integrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA; (2) small imperfect palindromes that are situated at the termini of the plasmid and are cognate with the active sites for plasmid integration into mtDNA; (3) two large, non-overlapping open-reading frames, ORF-1 and ORF-2, which are located on opposite strands of the plasmid and potentially encode RNA and DNA polymerases, respectively, and (4) a set of imperfect palindromes that coincide with similar structures that have been detected at more or less identical locations in the nucleotide sequences of other linear mitochondrial plasmids. The nucleotide sequence does not reveal a distinct gene that codes for the protein that is attached to the ends of the plasmid. However, a 335-amino acid, cryptic, N-terminal domain of the putative DNA polymerase might function as the terminal protein. Although the plasmid has been co-purified with nuclei and mitochondria, its nucleotide composition and codon usage indicate that it is a mitochondrial genetic element.

Differentiation of species and strains among filamentous fungi by DNA fingerprinting.

Abstract: We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)8, (GTG)5 and (GACA)4, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)4 produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.

Chromosome length polymorphisms in a Septoria tritici population

Abstract: Transverse alternating field electrophoresis (TAFE) was used to compare the electrophoretic karyotypes of seven Septoria tritici isolates sampled from a single population. Isolates were selected based on differences at 12 DNA restriction fragment length polymorphism (RFLP) loci. Significant differences in electrophoretic karyotype existed among the isolates. Isolates had 14–16 bands, believed to correspond to chromosomes, ranging in size from approximately 330 to 3500 kb. Homologous chromosomes were identified by hybridization of anonymous single-locus and multiplelocus nuclear DNA sequences to Southern blots of TAFE gels. Length differences of up to 20% existed among homologous chromosomes. Densitometry and probe hybridization data showed that several bands contained two chromosomes. Probe pSTS192 hybridized to two chromosomes in each isolate, supporting previous data which suggested that this probe hybridized to two unlinked RFLP loci. Hybridization with probe pSTL53 provided additional evidence of partial diploidy at an RFLP locus in one isolate.

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India.

Abstract: Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5′ termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.

Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells.

Abstract: The mutation pat1-114 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both pat1-114 and pat1+ diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the pat1-114-driven meiosis. Commitment to recombination and to meiosis have been timed at 2 h after meiotic induction. Due to a seven-fold reduction of intragenic recombination frequency in the ade6 region of pat1-114 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that influence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the pat1 allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed.

Identification and characterization of a second polygalacturonase gene of Aspergillus niger

Abstract: The filamentous fungus Aspergillus niger produces several endopolygalacturonases that are involved in the degradation of pectin. PGI, the enzyme representing the second most abundant activity in a commercial enzyme preparation, was further characterized and the corresponding gene was isolated. The nucleotide sequence of the pgaI gene was determined and the protein coding region was found to be interrupted by two short introns, one of which has a unusual donor splice site. The deduced 368 amino acids long protein with a putative prepropeptide of 31 amino acids shows 60% sequence identity to PGII in the mature protein. PGI overproducing A. niger strains were obtained by cotransformation with the cloned gene.

Plastid genome structure and plastid-related transcript levels in albino barley plants derived from another culture.

Abstract: Southern analysis of DNA from four albino barley plants regenerated from microspores by direct embryogenesis revealed the presence of plastid genomes which had undergone deletion or alteration of specific restriction fragments (delta ptDNAs). In contrast, a fifth plant appeared to contain an intact plastid genome. All the albino plants studied contained reduced amounts of ptDNA, the most abundant restriction fragments being present at levels between 6% and 20% of those found in the leaves of green seedlings. Steady-state levels of transcripts from plastid and nuclear genes encoding plastid components were estimated by Northern analysis of RNA from albino plants. Transcripts from the plastid genes rbcL, psbD-psbC and the 16S and 23S rRNAs were undetectable or were present at greatly reduced levels in albino plants compared to those found in green leaves. Transcripts from the nuclear genes rbcS and cab, which encode chloroplast localised proteins, were also present at reduced levels in albino pollen plants. Levels of the nuclear encoded 25S rRNA, which is not a plastid component, were found to be identical in albino plants and green leaves suggesting that only the expression of plastid-related genes may be affected in albino plants. The general reduction of plastid-related transcripts was independent of the different patterns of ptDNA alteration seen in albino pollen plants.

The SNQ3 gene of Saccharomyces cerevisiae confers hyper-resistance to several functionally unrelated chemicals.

Abstract: A multi-copy plasmid containing the SNQ3 gene confers hyper-resistance to 4-nitroquinoline-N-oxide (4NQO), Trenimon, MNNG, cycloheximide, and to sulfometuron methyl in yeast transformants. Restriction analysis, subcloning, and DNA sequencing revealed an open reading frame of 1,950 bp on the SNQ3-containing insert DNA. Gene disruption and transplacement into chromosomal DNA yielded 4NQO-sensitive null mutants which were also more sensitive than the wild-type to Trenimon, cycloheximide, sulfometuron methyl, and MNNG. Hydropathic analysis showed that the SNQ3-encoded protein is most likely not membrane-bound, while the codon bias index points to low expression of the gene.

Electrophoretic karyotyping of Leptosphaeria maculans differentiates highly virulent from weakly virulent isolates.

Abstract: Whole chromosomes from the fungal phytopathogen Leptosphaeria maculans were separated by transverse alternating field electrophoresis. The chromosome complements from several isolates of both highly and weakly virulent strains were compared. Small variations in chromosome size and apparent number were detected among isolates of the same strain. However, dramatic differences in both chromosome number and size were found when isolates of the highly virulen strain were compared to those of the weakly virulent. Highly virulent isolates had 6–8 distinct bands whereas weakly virulent isolates had 12–14. The genome sizes were estimated to be at least 8.6x106 base pairs for the virulent strain and 1.6x107 base pairs for the weakly virulent strain. The major differences found in the chromosome complements of the two strains, in combination with results of other biochemical, morphological, and genetic studies, indicate that they are distinct species.

Sequence analysis of wheat mitochondrial transcripts capped in vitro: definitive identification of transcription initiation sites.

Abstract: To identify transcription initiation sites in wheat mitochondria, the nascent 5′-ends of transcripts were specifically labeled by incubation of wheat mitochondrial RNA with [α-32P]GTP in the presence of the enzyme guanylyltransferase. After separation of the resulting capped transcripts by electrophoresis in polyacrylamide gels, individual RNAs were recovered and directly sequenced. Four RNA sequences obtained in this way were localized upstream of the protein-coding genes atpA, coxII, coxIII and orf25. Comparison of mRNA and gene sequences allowed precise positioning of transcription initiation sites for these four genes. Sequence similarities immediately upstream of these sites define a conserved motif that we suggest as a candidate regulatory element in wheat mtDNA. The relationship between this motif and putative mitochondrial promoters in other plant species is discussed.

Genomic organization and sequence analysis of the cytochrome oxidase subunit II gene from normal and male-sterile mitochondria in sugar beet

Abstract: We have cloned and sequenced the cytochrome oxidase subunit II (coxII) gene from both normal and cytoplasmic male-sterile (CMS) sugar beet. The normal coxII (designated NcoxII) locus was found to be located 1491 bp upstream from the gene for cytochrome oxidase subunit I (coxI) on the same DNA strand and to have a 1463 bp intron which split the coding sequence into two exons (382 and 398 bp). The COXII protein contains 260 amino acid residues. We have also found two copies of the coxII gene (ScoxII-1 and ScoxII-2) to be present in the CMS genome. Our results suggest that the NcoxII gene diverges completely from the ScoxII-1 and ScoxII-2 genes 50 bp 5′ to the ATG start codon. In addition, the ScoxII-1 and ScoxII-2 sequences could be readily discriminated from each other by the 3′ end and the immediately adjacent flanking sequences of the gene: the 3′ divergence results in a 101 codon extension of the ScoxII-2 ORF. Northern blot analysis demonstrates that the coxII gene exhibits altered transcript patterns in CMS compared with normal sugar beet. Different genomic arrangements of the coxII gene are considered to be the result of extensive intra-and inter-molecular recombination events involving the repeated DNA elements in the mitochondrial genome.

Nucleotide sequence of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase.

Abstract: The nucleotide sequence of the ERG12 gene, encoding mevalonate kinase, from Saccharomyces cerevisiae is presented. The longest open reading frame may code for a protein containing 443 amino acids with a deduced relative molecular mass of 48 500. The analysis of the nucleotide sequence reveals a complete identity with the yeast gene RAR1, isolated elsewhere by complementation of a rar1 mutation involved in the stability of plasmids with weak ARS. In addition, we show that mevalonate kinase is not a rate-limiting enzyme; however its sensitivity to FFP could be a key regulatory mechanism in the sterol pathway of yeast.

DNA fingerprinting of Ascochyta rabiei with synthetic oligodeoxynucleotides

Abstract: The ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea (Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA)4, (GTG)5, (CA)8, and (TCC)5, respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions.

Chimeric mitochondrial genes expressed in the C male-sterile cytoplasm of maize.

Abstract: Aberrant recombinations involving the mitochondrial atp9, atp6 and coxII genes have created unique chimeric sequences in the C male-sterile cytoplasm (cms-C) of maize. An apparent consequence of the rearrangements is the interchanging of transcriptional and/or translational regulatory signals for these genes, and alterations in the reading frames encoding the atp6 and coxII genes in the C cytoplasm. Particularly unusual is the organization of the atp6 gene in cms-C mitochondria, designated atp6-C. The atp6-C sequence is a triple gene fusion product comprised of DNAs derived from atp9, atp6 and an open reading frame of unknown origin. Although there is no direct evidence indicating that these chimeric genes are responsible for the cytoplasmic male sterility (cms) trait, their novel arrangements and the strong correlation between these genes and the C type of male sterility suggest such a role.

A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron.

Abstract: During interspecific crosses between Chlamydomonas eugametos and Chlamydomonas moewusii, an optional group I intron of 955 base pairs (CeLSU· 5) in the C. eugametos chloroplast large subunit rRNA gene undergoes a duplicative transposition event which is associated with frequent co-conversion of flanking cpDNA sequences. In the present study, we show that the basic protein of 218 amino acids encoded by CeLSU· 5 could mediate the phenomenon of intron transposition, also called intron homing. We overexpressed the ORF specifying this protein in E. coli using expression vectors that contain a C. moewusii cpDNA sequence encompassing the intron homing site. The expression product was found to exhibit a double-strand DNA endonuclease activity that is specific for the homing site. This activity was detected in vivo by self-linearization of the expression plasmids.

Distribution of RNA editing sites in Oenothera mitochondrial mRNAs and rRNAs.

Abstract: To investigate whether RNA editing in plant mitochondria modifies structural RNAs as well as protein-coding RNAs we compared the genomic-encoded information with the respective transcripts of several genes in Oenothera. The genes analysed are the 5S, 18S and 26 S rRNAs, the alpha-subunit of ATPase (atpA), cytochrome b (cytb), orfB, which is located upstream of cytochrome oxidase subunit III, and the respective leader, trailer and spacer sequences. All open reading frames were found to be edited to some degree. The atpA coding region has the least edited mRNA in Oenothera mitochondria, with only four nucleotides altered in the 1533 nucleotide open reading frame. From this analysis we conclude that frequent RNA editing is indicative of functional protein coding regions in plant mitochondria. The extensive editing in orfB, for example, suggests that this orf codes for a mitochondrial protein. No RNA editing event was found in the 5S rRNA or in the 1824 nucleotides analysed of the 18S rRNA, but two nucleotides were found to be altered in the 1970 nucleotides compared for the 26S rRNA. One nucleotide alteration has changed C to U, the other in reverse U to C. However, only one of five cDNA clones covering this region shows the modifications, similar to many silent editing events in open reading frames. RNA editing in the structural RNAs thus does not seem to be essential for their function in the mitochondrial ribosome.

Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis.

Abstract: Linear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae. Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5′ ends. The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination. Nuclear promoters are not recognised in the cytoplasm.

Polymorphic karyotypes in related Acremonium strains.

Abstract: A restriction fragment length polymorphism (RFLP) analysis was performed on six related Acremonium strains. With respect to the restriction fragment pattern, all strains of A. chrysogenum were indistinguishable from each other but showed distinctive differences from those of A. strictum, A. flavum and Cephalosporium polyvaleurum. Using pulsed-field gel electrophoresis, we obtained different chromosome patterns from most of the Acremonium strains. Remarkably, the pattern varies in three related A. chrysogenum strains which also differ in their rate of cephalosporin C biosynthesis. The electrophoretic karyotyping was confirmed by the location of rDNA genes on separate chromosomes. Our data indicate that chromosome translocations in industrial strains may be responsible for increased β-lactam synthesis.

A termination codon is created by RNA editing in the petunia mitochondrial atp9 gene transcript

Abstract: Analysis of the cDNA of the atp9-1 gene transcript from petunia mitochondria has revealed that ten C residues of the gene sequence are edited into U in the mRNA. Seven of these edits result in amino acid changes and one introduces a stop codon before the end of the open reading frame predicted from the gene sequence. The resulting protein is better conserved when compared to the same protein in other organism. Comparison of the edited petunia sequence with other plant mitochondrial atp9 gene sequences indicates variation in the number and positions of edits required to obtain the same amino acids in ATP9 polypeptides of higher plants.

Rubisco spacer sequence divergence in the rhodophyte alga Gracilaria verrucosa and closely related species.

Abstract: In the red alga Gracilaria verrucosa, the genes encoding the large and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are separated by a short spacer of less than 131 bp. Sequencing of PCR-amplified Rubisco spacers from a number of populations of G. verrucosa was performed to assess the feasibility of using this sequence for discriminating among closely related species or populations. Intrapopulation comparisons of the nucleotide sequences of these spacers from five isolates of G. verrucosa, and similar species, demonstrated four main groups. The first group included isolates from Europe and Argentina while the other groups are correlated with the geographical location of their origin.

Homology between mitochondrial DNA of Agaricus bisporus and an internal portion of a linear mitochondrial plasmid of Agaricus bitorquis.

Abstract: Agaricus bisporus, the cultivated mushroom, contains a mitochondrial fragment (50H) which was previously demonstrated by Southern hybridization to have sequence similarity to an internal region of pEM, a linear mitochondrial plasmid of Agaricus bitorquis. The nucleotide sequence of 50H was determined and compared to the sequence of the corresponding pEM fragment. The region of sequence homology on pEM is contained within an open reading frame (ORF) that may encode an RNA polymerase, but 50H is neither an intact nor a complete copy of the ORF. pEM also contains an ORF with characteristics of genes for virus-encoded DNA polymerases. pEM appears to be very similar to other linear mitochondrial plasmids (in fungi and higher plants) reported to contain ORFs that may encode the same types of polymerases. The potential functionality of the pEM sequence suggests that it has diverged less than the mitochondrial fragment from a common ancestor.

Isolation and primary structure of the ERG9 gene of Saccharomyces cerevisiae encoding squalene synthetase.

Abstract: The ERG9 gene of Saccharomyces cerevisiae has been cloned by complementation of the erg9-1 mutation which affects squalene synthetase. From the 5kkb insert isolated, the functional gene has been localized on a DNA fragment of 2.5 kb. The presence of squalene synthetase activity in E. coli bearing the yeast DNA fragment isolated, indicates that the structural gene encoding squalene synthetase has been cloned. The sequence of the 2.5 kb fragment contains an open reading frame which could encode a protein of 444 amino acids with a deduced relative molecular mass of 51 600. The amino acid sequence reveals one to four potential transmembrane domains with a hydrophobic segment in the C-terminal region. The N-terminus of the deduced protein strongly resembles the signal sequence of yeast invertase suggesting a specific mechanism of integration into the membranes of the endoplasmic reticulum.

PDC6, a weakly expressed pyruvate decarboxylase gene from yeast, is activated when fused spontaneously under the control of the PDC1 promoter.

Abstract: Three structural genes encode the pyruvate decarboxylase isoenzymes in the yeast Saccharomyces cerevisiae. PDC1 and PDC5 are active during glucose fermentation where PDC1 is expressed about six times more strongly than PDC5. Expression of PDC6 is weak and seems to be induced in ethanol medium. Consequently, pdc1 delta pdc5 delta double mutants do not ferment glucose and do not grow on glucose medium. Spontaneous mutants, derived from such a pdc1 pdc5 strain, were isolated which could again ferment glucose. They showed pyruvate decarboxylase activity due to a duplication of PDC6. The second copy of PDC6 was expressed under the control of the PDC1 promoter, which was still present in the pdc1 strain. However, the resulting PDC1-PDC6 fusion gene could only partially substitute for PDC1: to achieve normal growth and high pyruvate decarboxylase activity strains carrying PDC1-PDC6 required a functional PDC5 gene which is dispensable in a PDC1 wild-type background. Thus, expression of PDC5 depends on the state of the PDC1 locus: low in the PDC1 wild-type background and high in PDC1-PDC6 fusion strains and, as shown previously, in pdc1 mutants. The activation of PDC5 expression in PDC1-PDC6 strains may be due to particular properties of the PDC1-PDC6 fusion protein or simply to the weaker expression of PDC1-PDC6 in comparison to the wild-type PDC1 gene.