Showing papers in "Current Genetics in 1987"

Sequence analysis of cDNAs for the human and bovine ATP synthase β subunit: mitochondrial DNA genes sustain seventeen times more mutations

Abstract: We have cloned and sequenced human and bovine cDNAs for the β subunit of the ATP synthase (ATP-syns), a nuclear DNA (nDNA) encoded oxidative phosphorylation (OXPHOS) gene. The two cDNAs were found to share 99% amino acid homology and 94% nucleotide homology. The evolutionary rate of ATPsyns was then compared with that of two mitochondrial DNA (mtDNA) ATP synthase genes (ATPase 6 and 8), seven other mtDNA OXPHOS genes, and a number of nuclear genes. The synonymous substitution rate for ATPsyns proved to be 1.9 × 10−9 substitutions per site per year (substitutions × site−1 × year−1) (SSY). This is less than 1/2 that of the average nDNA gene, 1/12 the rate of ATPase 6 and 8, and 1/17 the rate of the average mtDNA gene. The synonymous and replacement substitution rates were used to calculate a new parameter, the “selective constraint ratio”. This revealed that even the most variable mtDNA protein was more constrained than the average nDNA protein. Thus, the high substitution mutation rate and strong selective constraints of mammalian mtDNA proteins suggest that mtDNA mutations may result in a disproportionately large number of human hereditary diseases of OXPHOS.

Chloroplast DNA from lettuce and Barnadesia (Asteraceae): structure, gene localization, and characterization of a large inversion

Abstract: We have cloned into plasmids 17 of 18 lettuce chloroplast DNA SacI fragments covering 96% of the genome. The cloned fragments were used to construct cleavage maps for 10 restriction enzymes for the chloroplast genomes of lettuce (Lactuca sativa) and Barnadesia caryophylla, two distantly related species in the sunflower family (Asteraceae). Both genomes are approximately 151 kb in size and contain a 25 kb inverted repeat. We also mapped the position and orientation of 37 chloroplast DNA genes. The mapping studies reveal that chloroplast DNAs of lettuce and Barnadesia differ by a 22 kb inversion in the large single copy region. Barnadesia has retained the primitive land plant genome arrangement, while the inversion has occurred in a lettuce lineage. The endpoints of the derived lettuce inversion were located by comparison to the well-characterized spinach and tobacco genomes. Both endpoints are located in intergenic spacers within tRNA gene clusters; one cluster being located downstream from the atpA gene and the other upstream from the psbD gene. The endpoint near the atpA gene is very close to one endpoint of a 20 kb inversion in wheat (Howe et al. 1983; Quigley and Weil 1985). Comparison of the restriction site maps gives an estimated sequence divergence of 3.7% for the lettuce and Barnadesia genomes. This value is relatively low compared to previous estimates for other angiosperm groups, suggesting a high degree of sequence conservation in the Asteraceae.

Chloroplast DNA evolution among legumes: Loss of a large inverted repeat occurred prior to other sequence rearrangements

Abstract: We have compared the sequence organization of four previously uncharacterized legume chloroplast DNAs - from alfalfa, lupine, wisteria and subclover — to that of legume chloroplast DNAs that either retain a large, ribosomal RNA-encoding inverted repeat (mung bean) or have deleted one half of this repeat (broad bean). The circular, 126 kilobase pair (kb) alfalfa chloroplast genome, like those of broad bean and pea, lacks any detectable repeated sequences and contains only a single set of ribosomal RNA genes. However, in contrast to broad bean and pea, alfalfa chloroplast DNA is unrearranged (except for the deletion of one segment of the inverted repeat) relative to chloroplast DNA from mung bean. Together with other findings reported here, these results allow us to determine which of the four possible inverted repeat configurations was deleted in the alfalfa-pea-broad bean lineage, and to show how the present-day broad bean genome may have been derived from an alfalfa-like ancestral genome by two major sequence inversions. The 147 kb lupine chloroplast genome contains a 22 kb inverted repeat and has essentially complete colinearity with the mung bean genome. In contrast, the 130 kb wisteria genome has deleted one half of the inverted repeat and appears colinear with the alfalfa genome. The 140 kb subclover genome has been extensively rearranged and contains a family of at least five dispersed repetitive sequence elements, each several hundred by in size; this is the first report of dispersed repeats of this size in a land plant chloroplast genome. We conclude that the inverted repeat has been lost only once among legumes and that this loss occurred prior to all the other rearrangements observed in subclover, broad bean and pea. Of those lineages that lack the inverted repeat, some are stable and unrearranged, other have undergone a moderate amount of rearrangement, while still others have sustained a complex series of rearrangement either with or without major sequence duplications and transpositions.

The chloroplast tRNALys(UUU) gene from mustard (Sinapis alba) contains a class II intron potentially coding for a maturase-related polypeptide.

Abstract: The trnK gene endocing the tRNALys (UUU) has been located on mustard (Sinapis alba) chloroplast DNA, 263 by upstream of the psbA gene on the same strand. The nucleotide sequence of the trnK gene and its flanking regions as well as the putative transcription start and termination sites are shown. The 5′ end of the transcript lies 121 by upstream of the 5′ tRNA coding region and is preceded by procaryotic-type “−10” and “−35” sequence elements, while the 3′ end maps 2.77 kb downstream to a DNA region with possible stem-loop secondary structure. The anticodon loop of the tRNALys is interrupted by a 2,574 by intron containing a long open reading frame, which codes for 524 amino acids. Based on conserved stem and loop structures, this intron has characteristic features of a class II intron. A region near the carboxyl terminus of the derived polypeptide appears structurally related to maturases.

Transformation of Aspergillus niger using the homologous orotidine-5'-phosphate-decarboxylase gene.

Abstract: A homologous transformation system for the filamentous fungus Aspergillus niger has been developed, based on the orotidine-5′-phosphate-decarboxylase gene. A. niger Pyr− mutants have been selected from 5-fluoroorotic acid resistant mutants. These mutants were found to comprise two complementation groups, pyrA and pyrB. The A. niger OMP-decarboxylase gene was isolated from a gene library by heterologous hybridization with the Neurospora crassa pyr4 gene. The cloned gene is capable to transform A. nidulans pyrG mutants at high frequencies. Transformation of A. niger pyrA mutants occurs with moderate frequencies (about 50 transformants/μg DNA) whereas the pyrB mutants cannot be complemented with the cloned OMP-decarboxylase gene. Analysis of the DNA of the A. niger PyrA+ transformants showed that transformation resulted in integration of the vector DNA into the genome by homologous recombination. Both gene replacements and integration of one or more copies of the complete vector have been observed.

Unicircular structure of the Brassica hirta mitochondrial genome.

Abstract: Restriction mapping studies reveal that the mitochondrial genome of white mustard (Brassica hirta) exists in the form of a single circular 208 kb chromosome. The B. hirta genome has only one copy of the two sequences which, in several related Brassica species, are duplicated and undergo intramolecular recombination. This first report of a plant mitochondrial DNA that does not exist in a multipartite structure indicates that high frequency intramolecular recombination is not an obligatory feature of plant mitochondrial genomes. Heterologous filter hybridizatios reveal that the mitochondrial genomes of B. hirta and B. campestris have diverged radically in sequence arrangement, as the result of approximately 10 large inversions. At the same time, however, the two genomes are similar in size, sequence content, and primary sequence.

Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pKD1

Abstract: The circular plasmid pKD1 (or 1.6 μm DNA) has recently been isolated from Kluyveromyces drosophilarum. This plasmid appears to have a functional organization analogous to that of the 2 μ DNA of Saccharomyces cerevisiae, although the respective nucleotide sequences show little homology. pKD1 can be transferred to Kluyveromyces lactis where it is replicated stably. Using recombinant molecules derived from pKDl, a practical transformation system has been developed for Kluyveromyces lactis, with an efficiency and stability comparable to the 2 μ-based Saccharomyces cerevisiae transformation system.

Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences

Abstract: In order to probe the physiological role of the yeast plasma membrane ATPase we have replaced the constitutive promoter of its gene by a galactose-dependent promoter. The resulting cells stop growing on glucose medium when the preformed ATPase is diluted to 20% of normal. There is a correlation between ATPase activity and both proton efflux from the cells and amino acid transport. A large proportion of growth-arrested cells appear enlarged and with several buds containing nuclei.

LEU2 directed expression of beta-galactosidase activity and phleomycin resistance in Yarrowia lipolytica.

Abstract: The nucleotide sequence of a 968 by DNA fragment spanning the promoter and the 5′ upstream sequence of the LEU2 coding sequence of the yeast Yarrowia lipolytica has been determined. A LEU2:lacZ gene fusion has been constructed and expressed in transformed yeast cells, showing that as few as 232 by of the LEU2 promotor were sufficient to direct gene expression. In order to develop new markers for transformation of this yeast, the LEU2 initiation codon was destroyed by in vitro mutagenesis and replaced by a cloning site. A gene confering phleomycin resistance in E. coli was attached to the LEU2 promoter and shown to be efficiently expressed in yeast: direct selection of phleomycin resistant transformants was possible.

The maize plastid psbB-psbF-petB-petD gene cluster: spliced and unspliced petB and petD RNAs encode alternative products.

Abstract: The chloroplast psbB, psbF, petB, and petD genes are cotranscribed and give rise to many overlapping RNAs. The mechanism and significance of this mode of expression are of interest, particularly because the accumulation of the psb and pet gene products respond differently to both light and, in C4 species such as maize, developmental signals. We present an analysis of the maize psbB, psbF, petB, and petD genes and intergenic regions. The genes are organized similarly in maize (a C4 species) and in several C3 species. Functional class II-like introns interrupt the 5′ ends of petB and petD. Both spliced and unspliced RNAs accumulate; these encode alternative forms of the petB and petD proteins, differing at their N-termini. Promoter-like elements between psbF and petB, and biased codon usage suggest that the differential regulation of the psb and pet genes might be achieved at both the transcriptional and translational levels.

Transformation of Fulvia fulva, a fungal pathogen of tomato to Hygromycin B resistance

Abstract: A transformation system for the tomato pathogen Fulvia fulva has been developed. Hygromycin B resistant colonies were obtained after treatment of protoplasts with a plasmid containing an E. coli hygromycin B phosphotransferase gene fused to an Aspergillus nidulans promoter. The DNA was stably integrated into the genome. The number and sites of integrations varied among transformants. The demonstration of transformation opens the way for the molecular genetic analysis of the interaction of Fulvia with tomato.

The gene for the 9 kd polypeptide, a possible apoprotein for the iron-sulfur centers A and B of the photosystem I complex, in tobacco chloroplast DNA

Abstract: The gene for the 9 kd polypeptide (a possible apoprotein for the iron-sulfur centers A and B) of photosystem I has been located in the small single-copy region of tobacco chloroplast DNA. This gene (psaC) was identified by comparing the N-terminal amino acid sequence of the spinach 9 kd polypeptide with the entire sequence of tobacco chloroplast genome. The gene organization is ndhE (101 codons)--263 bp spacer--psaC (81 codons)--94 bp spacer--ndhD (509 codons). Northern blot hybridization revealed that psaC is transcribed in the chloroplasts. The deduced amino acid sequence and secondary structure are presented. The predicted polypeptide is rich in cysteine residues and contains a unique repeated sequence.

Efficient integrative transformation of Cephalosporium acremonium.

Abstract: A hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 bp sequence of Cephalosporium acremonium DNA next to the 5' end of a bacterial open reading frame, HPTorf. The sequence was obtained as an 850 bp NcoI restriction fragment from the 5' non-coding region of the C. acremonium isopenicillin N synthetase (IPNS) gene. The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT). Plasmids that contained IPNSp/HPTorf transformed C. acremonium to a stably maintained hygromycin B resistant phenotype. Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants. The number of transformants per microgram of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence. Plasmids with the promoterless HPTorf and plasmids with a truncated S. cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C. acremonium at equivalent low frequencies. Transformation of C. acremonium with linearized plasmid DNA produced at least 2-3 fold more transformants than the corresponding circular molecule. Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells. Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C. acremonium ribosomal DNA (rDNA), or a fragment of C. acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S. cerevisiae, or putative transcriptional termination/polyadenylation signals from the IPNS gene. These plasmids transformed C. acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements.

Genetic nomenclature and gene list of the fission yeast Schizosaccharomyces pombe

Abstract: The nomenclature rules for the genetics of the fission yeast Schizosaccharomyces pombe have been fixed for the first time, after discussion among scientists working with this organism. Conventions are proposed for the naming of genes and alleles that are obtained by classical means or by reverse genetics. In addition a list has been compiled of 460 known genes of S. pombe. It includes genes defined both by classical mutation analysis and by molecular cloning. 270 genes have been assigned either to one of the three nuclear chromosomes or the mitochondrial genome.

Carotenoid mutants of Gibberella fujikuroi

Abstract: The orange pigment neurosporaxanthin colours the mycelia of wild Gibberella fujijuori (Fusarium monifliforme) grown in the light, but is barely detectable in the dark. We have isolated carotenoid mutants from conidia exposed to N-methyl-N′-nitro-N-nitroso-guanidine and other mutagens. Specific blocks in the pathway are represented by white mutants accumulating phytoene and red mutants accumulating torulene; there are also mutants without carotenoids or with complex carotenoid mixtures. Regulatory mutants overproduce neurosporaxanthin, both in the light and in the dark. Other mutants contain considerable neurosporaxanthin in the dark, but less than in the light. The results bring out similarities between the carotenoid biosynthetic pathways of Gibberella and Phycomyces, and significant differences in their respective regulations.

Transformation of Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker.

Abstract: The Aspergillus nidulans acetamidase gene (amdS) has been used to transform Penicillium chrysogenum at low frequency Several transformants were tested and shown to be mitotically stable Southern blot analysis indicated that transforming DNA had integrated into the chromosomal DNA, possibly at multiple sites

Selection and characterization of pyrG mutants of Penicillium chrysogenum lacking orotidine-5′-phosphate decarboxylase and complementation by the pyr4 gene of Neurospora crassaa

Abstract: Pyrimidine auxotrophs of Penicillium chrysogenum have been isolated at a high frequency among mutants resistant to 5-fluoroorotic acid (5.2 mM). Some of the pyrimidine auxotrophs (e.g. strain pyrG1) showed no reversion. A radiometric assay based on the conversion of (6-14C)orotidine 5′-monophosphate (OMP) into (6-14C)uridine 5′-monophosphate (UMP) was developed to determine OMP-decarboxylase activity. One of the pyrimidine auxotrophs (P. chrysogenum pyrGl) was studied in detail. It was deficient in OMP-decarboxylase activity, whereas the parental strain (P. chrysogenum Wis. 54-1255) showed a normal enzyme activity. A five-fold higher OMP-decarboxylase activity was found in a P. chrysogenum pyrGI clone transformed with plasmids containing the Neurospora crassa pyr4 gene (which codes for the same enzyme).

Characterization of a chloroplast mutation in the psaA2 gene of Chlamydomonas reinhardtii

Abstract: The synthesis of polypeptides related to the CPI chlorophyll-protein complex of photosystem I has been studied by pulse-labeling experiments in twenty chloroplast mutants of Chlamydomonas reinhardtii. Three mutations of the same locus (Girard-Bascou 1987) result in the absence of these CPI-related polypeptides. Among these mutations one, (FUD26) leads to the synthesis of a new polypeptide presumed to be a truncated CPI apoprotein. The molecular characterization of this mutation in the psaA2 gene has been achieved by DNA sequencing the 3′ end of this gene. The FUD26 mutation is a 4 base pair deletion resulting in frameshift and premature termination of the protein.

Multiple copies of the amdS gene of Aspergillus nidulans cause titration of trans-acting regulatory proteins

Abstract: It has been established that a plasmid containing the amdS gene of Aspergillus nidulans may be used to transform amdS+ strains by selecting for increased utilization of acetamide as sole nitrogen source. Analysis of transformants has shown that multiple tandem copies of the plasmid can be integrated into the chromosome, commonly at sites other than the amdS locus. While the transformed phenotype was relatively stable through mitotic and meiotic divisions evidence was found for variation in plasmid copy number presumably due to unequal recombination events. Expression of the integrated amdS genes was related to copy number, and the amdS RNA produced was similar in size to wild-type RNA. Evidence for titration of the product of the regulatory gene amdR by multiple copies of amdS was found. No titration of the product of the areA gene was observed, and amdS expression was still dependent on areA function. Multiple copies of the amdI9 mutation resulted in poor growth on acetate. This was not observed in the case of the amdS+ gene. The cis-acting amdI9 mutation causes increased facB dependent acetate induction of amdS expression. Titration of the facB gene produce by amdI9 DNA, but not by amdS+ DNA, therefore suggested that the mutation results in increased affinity for the facB gene product.

Regulation of the trehalose-6-phosphate synthase complex in Saccharomyces. I. Interconversion of forms by phosphorylation.

Abstract: Trehalose-6-phosphate synthase is another example of an enzyme of carbohydrate metabolism, in Saccharomyces, which could be regulated by interconversion of forms. Deactivation was mediated both in vivo and in vitro by a cyclic AMP-dependent protein kinase. Reversibility of this process was obtained by a phosphatase treatment leading to an increase in activity. The phosphorylated, less active form of the enzyme proved to be more susceptible to activation by ATP.Mg. Mutants with well defined lesions in the cyclic AMP-dependent protein kinase system were used to corroborate our findings of a possible regulatory mechanism of trehalose-6-phosphate synthase activity by interconversion of forms.

Sex appeal in fission yeast

Abstract: Using diploid strains of Schizosaccharomyces pombe, cytological evidence has been obtained which shows that not only h− cells (Fukui et al. 1986) but also h+ cells secrete a diffusible mating pheromone that attracts cells of the opposite mating type.

Extensive mitochondrial DNA variation in somatic tissue cultures initiated from wheat immature embryos

Abstract: Wheat mitochondria) DNA has been isolated from callus cultures initiated from both immature embryos and the corresponding parental cultivar. A Sall restriction pattern study has shown that the organization of callus culture mitochondria) DNA underwent extensive change, characterized by either the disappearance or the decrease in the relative stoichiometry of several restriction bands. Hybridization of labelled mitochondrial fragments obtained from a recombinant cosmid library to Southern blots of callus and parental line restricted mitochondria) DNAs has shown that a fraction of the mitochondria) genome was lost in callus cultures. Data from a Sall + HindIII restriction map of a defined part of the wheat mitochondria) genome concerned with some of these variations strongly suggest that the observed variations correspond to the disappearance of at least one mitochondria) DNA subgenomic molecule in callus cultures.

Cell lineage asymmetry in Schizosaccharomyces pombe: unilateral transmission of a high-frequency state for mating-type switching in diploid pedigrees

Abstract: Sporulation was followed in diploid pedigrees of the fission yeast Schizosacharomyces pombe of the genotype h 90 //h −U . From the pattern of dividing cells and asci over 6–8 cell cyles information was gathered on the underlying laws regulating mating-type switching in this year. The results are discussed in relation to the introduction and transmission of double-strand breaks at the smt signal dose to the mat1 cassette. Such cuts have previously been reported as being a prerequisite for mating-type switching. The general criteria appear as follows. Each cell giving rise to a switch carries a cut at smt. A newly switched cassette is not associated with a cut. The sister cell to a newly switched cell retains bot the original cassette and the double-strand cut. Most of the time a “virgin” cell without the cut divides unequally so that one and only one of the daugthers obtains a cut. The consequence of these criteria is that long series of switch after switch in the same direction (“recurrent swtiching”) were observed in the pedigrees.

Construction of brewer's yeasts secreting fungal endo-ß-glucanase

Abstract: Barley β-glucans present in wort reduce beer filtrability and cause hazes and precipitates in the finished beer The endo-β-1,4-glucanase enzyme, EGI, found in the filamentous fungus Trichoderma reesei, is capable of efficiently hydrolyzing these β-glucans The cDNA copy of the eg11 gene, which codes for the EGI enzyme, was coupled to yeast regulatory sequences and transferred to a brewer's yeast using the yeast copper chelatin gene CUP1 as a selection marker in the transformation The eg11 gene was transferred to the yeast both on a multicopy plasmid and on an integrating plasmid In both cases, highly glycosylated, active EGI enzyme was secreted into the medium Barley β-glucans present in wort were efficiently hydrolyzed by the recombinant brewer's yeast

Homology in the region containing a tRNATrp gene and a (complete or partial) tRNAPro gene in wheat mitochondrial and chloroplast genomes

Abstract: We have used bean mitochondrial (mt) and chloroplast (cp) tRNATrp as probes to locate the corresponding genes on the mt and cp genomes of wheat and we have determined the nucleotide sequences of the wheat mt and cp tRNATrp genes and of the flanking regions. Sequence comparisons show that the wheat mt and cp tRNATrp genes are 97% homologous.

Cloning and expression of the OMP decarboxylase gene URA4 from Schizosaccharomyces pombe.

Abstract: URA4, the gene coding for orotidine monophosphate decarboxylase (OMPdecase), has been cloned from the fission yeast by homologous complementation and restricted in an Escherichia coli-Schizosaccharomyces pombe (E. coli-S. pombe) replicative plasmid to a 1.76 kb HindIII fragment. This plasmid is maintained at a high copy number in S. pombe and allows OMPdecase expression in Saccharomyces cerevisiae (S. cerevisiae) as well as in E. coli. After characterisation by restriction mapping and Southern hybridisation, the cloned gene was used as a probe to measure URA4 transcription and to examine its regulation. Messenger RNA levels were measured by DNA/RNA filter-hybridisation with pulse labelled RNAs during 6-azauridine (6-AUR) inhibited growth in wild type and 6-AUR sensitive strains. We found that in S. pombe the OMP analogue 6-AUR does not regulate the level of OMPdecase formation as it does in S. cerevisiae but rather modifies the ratio of total polyA+ to polyA− RNAs in the cell. Based on these results and on corresponding enzyme activities this study demonstrates divergent pyrimidine pathway regulation in the two yeasts S. cerevisiae and S. pombe. Finally, we propose the use of the URA4 gene as a convenient selective marker for genetic engineering in S. pombe.

The complex Arom locus of Aspergillus nidulans. Evidence for multiple gene fusions and convergent evolution.

Abstract: The physical positions of the DNA sequences encoding the five consecutive enzyme activities required to metabolise 3-deoxy-D-arabino-heptulosonic acid-7-phosphate to 5-enolpyruvyl-shikimate-3phosphate, which are encoded by the A. nidulans Arom polypeptide have been determined. Subfragments of the Arom locus encoding EPSP synthase and 3-dehydroquinase have been expressed in appropriate E. coli aro mutants. The DNA sequence of the A. nidulans Arom locus has been shown to have homology with the corresponding unlinked E. coli aro loci strongly suggesting (1) divergent evolution from common ancestral sequences and (11) that the complex A. nidulans Arom locus arose by multiple gene fusion. The DNA and protein sequence of the two 3-dehydroquinase isoenzymes of A. nidulans share no homology, strongly indicating separate phylogenetic origins and their convergent evolution. The 5′ and 3′ non-translated DNA sequence of the A. nidulans Arom locus is presented along with the presumed sites for transcription initiation and polyadenylation determined by S1 nuclease protection experiments.

Mitochondrial plasmid DNAs of broad bean: nucleotide sequences, complex secondary structures, and transcription.

Abstract: Three circular plasmid DNA molecules of 1704, 1695 and 1476 nucleotide pairs from broad bean mitochondria (mt-plasmids 1–3) have been sequenced. Within a highly homologous segment of mt-plasmid 1 and 2 are found a series of six directly repeated, inverted repeat sequences, separated by unique sequences. Mt-plasmid 3 contains a series of four inverted repeat sequences, unrelated to the inverted repeat sequences of mt-plamids 1 and 2. Two RNA molecules of about 440 and 320 nucleotides that are complementary to mt-plasmid 2 were detected. Mapping of 5′ and 3′ termini of these complementary RNA molecules indicated that all transcription from mt-plasmid 2 occurs within a 441 nucleotide region of the molecule. Evidence for transcription of mt-plasmids 1 and 3 was not found.

Neomycin resistance as a dominantly selectable marker for transformation of the zygomycete Absidia glauca.

Abstract: A plasmid (pAmN61) containing the NPT II structural gene (neomycin phosphotransferase) fused to the N-terminal region of a homologous actin gene was used for the transformation of Absidia glauca protoplasts. Neomycin resistant transformants could be selected for on complete medium containing 1.2 mg/ml neomycin sulfate. The physical presence of plasmid DNA in Absidia glauca was demonstrated by retransformation into Escherichia coli and by Southern blot analysis. No integration of plasmid DNA at either one of the two actin loci was observed; Southern blot experiments provide evidence that pAmN61 is autonomously replicated in Absidia glauca.

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae: multiple trans-acting nuclear genes exert specific effects on expression of each of the cytochrome c oxidase subunits encoded on mitochondrial DNA

Abstract: Fourteen nuclear complementation groups of mutants that specifically affect the three mitochondrially-encoded subunits of yeast cytochrome c oxidase have been characterized. Genes represented by these complementation groups are not required for mitochondrial transcription, transcript processing, or translation per se but are required for the expression of one of the three genes — COX1, COX2, or COX3 — which encode the cytochrome c oxicase subunits I, II, or III, respectively. Five of these genes affect the biogenesis of cytochrome c oxidase subunit I, 3 affect the biogenesis of subunit II, 3 affect the biogenesis of subunit III and 3 affect the biogenesis of both cytochrome c oxidase subunit I and cytochrome b, the product of COB. Among the 5 complementation groups of mutants that affect the expression of COX1, 2 lack COX1 transcripts, 1 produces incompletely processed COX1 transcripts, and 2 contain normal levels of normal-sized COX1 transcripts. In contrast, all 3 complementation groups which affect the expression of COX2 and all 3 complementation groups which affect the expression of COX3 exhibit no, or little, detectable difference with respect to the wild type pattern of transcripts. The 3 complementation groups which affect the expression of both COX1 and COB all have aberrant COX1 and COB transcript patterns. These findings indicate that multiple trans-acting nuclear genes are required for specific expression of each COX gene encoded on mitochondrial DNA and suggest that their products act at different steps in the expression of these mitochondrial genes.