Abstract: A 1.6 kbp DNA segment of spinach plastid DNA has been shown to carry the gene for the proteolipid subunit of the ATP synthase. Each plastid chromosome contains one copy of this gene which is located in the large single-copy region of the chromosome near that of the ATP synthase alpha subunit. These two genes are transcribed in the same direction and probably in distinct RNA species. The proteolipid gene was located by hybrid-selection mapping, by transcription/translation of recombinant DNAs and by nucleotide sequencing. The in vitro product was identified by electrophoretic criteria including its characteristic shift in electrophoretic mobility upon incubation with dicyclohexylcarbodiimide, and immunology. The nucleotide sequence of the proteolipid gene is uninterrupted. The deduced amino acid sequence coincides with the published amino acid sequence for this protein and shows little homology with the published sequence of the proteolipid subunit of E. coli.

Abstract: Two types of DNA sequence polymorphisms were found among different geographic isolates of the basidiomycete fungus Coprinus einereus. These strains showed gains and losses of restriction enzyme recognition sites as well as extensive insertion/ deletion variation within DNA sequences present in a single copy per haploid genome. The same types of DNA sequence variants were also found within the tandemly repeated ribosomal RNA genes in these strains. There appears to be very little interspersed repetitive DNA in Coprinus, since all the randomly selected cloned DNA sequences studied in this survey were present only once in each haploid genome.

Abstract: Three sets of mitotic chromosomes were observed in wild type or cdc mutants (nda3-KM311 and nda2-KM52) of the fission yeast S. pombe by the DAPI staining method. The block of microtubular functions by thiabendazole or by the mutations caused their individual appearance in mitotically arrested cells. The chromosomes have a characteristic size; the length ratio of short, medium and long ones was roughly 1:2:3, consistent with the previous genetical data (Kohli et al. 1977). Double staining with ethidium bromide and DAPI showed that the nucleolus was always associated with the shortest chromosome. Pair-like structures resembling sister chromatids were also seen.

Abstract: The RAD52 gene of Saccharomyces cerevisiae has previously been shown to be involved in both recombination and DNA repair. Here we report on the cloning of this gene. A plasmid containing a 5.9 kb yeast DNA fragment inserted into the BamH1 site of the YEp13 vector has been isolated and shown to complement the X-ray sensitive phenotype of the rad52-1 mutation. The rad52-1 cells containing the plasmid form larger colonies than similar cells having lost the plasmid. This plasmid has been shown not to complement either the U.V. sensitivity or the recombination defect of the E. coli recA mutation. From the insert various fragments have been subcloned into the YRp7 and YIp5 vectors. Integration events of two of the subclones have been genetically mapped to the chromosomal location of RAD52, indicating that the structural gene has been cloned. A 1.97 kb BamH1 fragment subcloned into YRp7 in one orientation complements the rad52-1 mutation, while the same fragment in the opposite orientation fails to complement. Various other subclones indicate that a BglII site, within the BamH1 fragment, is in the RAD52 gene. This BglII site has been deleted by Sl-nuclease digestion and the resulting deletion inactivates the RAD52 gene. BAL31 deletions from one end of a 1.9 kb Sal1-BamH1 fragment have been isolated; up to 0.9 kb can be deleted without loss of RAD52 activity, indicating that the RAD52 gene is approximately 1 kb or less in length.

Abstract: The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells Recombination of mitochondrial genomes is not, however, precluded during dikaryosis Using monokaryons with different mitochondrial gene mutations, [acu-10] causing cytochrome aa3 deficiency and[cap-11] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons

Abstract: We have located the positions of the genes coding for the α, β and e subunits of the ATPase complex on Spirodela oligorhiza chloroplast DNA by means of heterologous hybridization with Spinacia cpDNA fragments.The overall cpDNA sequence organization of Petunia hybrida and Spirodela was compared. We hybridized well-characterized, cloned Spirodela cpDNA fragments with size fractionated Petunia cpDNA digested by Sall. It appears that the monocotyledonous Spirodela and the dicotyledonous Petunia cpDNA share a common sequence organization around their entire circumference. These observations, together with data reported in the literature, indicate a strikingly similar genetic organization of the chloroplast genome in widely divergent plants.

Abstract: Genetic analysis of crosses carried out between starch utilizing strains of Saccharomyces diastaticus and laboratory strains of S. cerevisiae has revealed the existence of a gene which inhibits the expression of the amylolytic capability in the resulting hybrids, as well as in the meiotic offspring of the crosses. This gene is unlinked to any of the three STA genes which are known to be responsible for starch utilization by S. diastaticus.

Abstract: Plasmids that complement the yeast mutations rad50-1, rad51-1, rad54-3 and rad55-3 were obtained by transforming strains that carried a leu2 marker and the particular rad mutation, with YEp13 plasmids containing near random yeast DNA inserts. Integration of these plasmids or of fragments of these plasmids was accomplished. Genetic studies using the integrants established the presence of the genes RAD51, RAD54 and RAD55 in the respective plasmids. However, a BamHI subclone of the rad50-1 complementing plasmid failed to integrate at the RAD50 locus, indicating that no homology exists between this fragment and the RAD50 gene.A BamHI fragment from the RAD54 plasmid was shown to be internal to the RAD54 gene: its integration within a wild type copy of RAD54 causes the cell to become Rad(-); its excision is X-ray inducible and restores the Rad(+) phenotype. Since cells bearing a disrupted copy of RAD54 are able to survive, we conclude that this gene is not essential.

Abstract: Considerable DNA sequence homology can be detected between the Escherichia coli genes coding for translational and transcriptional components and both the chloroplast and nuclear genomes of Chlamydomonas reinhardi. Labeled chloroplast DNA was demonstrated to hybridize to DNA fragments of the transducing phages λfus3 and λspc2 that encode ribosomal proteins of the α and S10 operons. Further, chloroplast DNA probes hybridize to fragments of λrtfd 18 that encode the β and β′ subunits of RNA polymerase. The regions homologous to the ribosomal protein and RNA polymerase genes were located on the chloroplast DNA physical map by probing restriction fragments of chloroplast DNA with phage or plasmid fragments carrying these E. coli genes. Probing nuclear DNA with bacterial gene probes revealed DNA fragments homologous to elongation factor and ribosomal protein genes. Most surprisingly, sequences homologous to the β subunit of RNA polymerase were found not only in chloroplast DNA but in nuclear DNA as well.

Abstract: The in vitro transfer of DNA, was mainly developed by using bacteria as host cells (Bernard and Helinski 1980). Accordingly, suitable vector systems for recombinant DNA technology are predominantly of prokaryotic origin. Since gene cloning is becoming a more essential component of both fundamental research (e.g. gene expression, regulation) (Losson and Lacroute 1981) and applied research (e.g. production of therapeutants and other products relevant for biotechnology) (Johnson and Burnett 1978), the inclusion of eukaryotes into the spectrum of host organisms becomes necessary. One of the main reasons to explore eukaryotes for such research is that bacteria, when used as hosts for cloning of eukaryotic DNA, may cause difficulties, such as failure in replication or expression of the foreign DNA; complications when extracting the product; or instability of the transformants (MacLeod 1980). In order to avoid complications of this kind, it is desirable for the cloning of eukaryotic DNA to be restricted to eukaryotic systems only, i.e. eukaryotes providing both the host and the vector. This concept requires:

Abstract: By using the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI) to stain DNA one can follow the pattern of events affecting plastid DNA which occur in the formation and maturation of individual zygotes of the green flagellate Chlamydomonas moewusii. This species, like C. reinhardi, expresses uniparental inheritance of plastid DNA characters among zygote progeny, and is particularly favorable for cytological observation because the locale of the contribution of each gamete can still be recognized in mature zygotes. Gametes contribute equal numbers of DNA nucleoids, and amounts of plastid DNA (as measured by DAPI-DNA micro spectrofluorometry), to the zygote at fusion. Starting at nine hours, coincident with the further fusion of cell contents, plastid DNA disappears from the plastid contributed by one gamete. Further slow coalescence of nucleoids leads to a final nucleoid number per zygote approximately 1/3 of the sum of the 2 gametes.The DNA loss from one gamete plastid may require plastid contact to be initiated. Both light and nutrient availability affect the final number and distribution of plastid DNA nucleoids in the mature zygote. These observations are related to known genetic and biochemical data on uniparental inheritance of plastid characters.

Abstract: U. maydis has been transformed to aminoglycoside antibiotic resistance by plasmid pMP81 DNA, which contains the yeast LEU-2 gene and 2-micron DNA inserted into pCRI, encoding a gene for an aminoglycoside phosphotransferase. The resistant phenotype of transformants is mitotically unstable in the absence of antibiotics. Closed, circular pMP81 DNA was detected in transformant DNA preparations by hybridization to pCR1 DNA and by transformation of Escherichia coli to kanamycin resistance. Plasmid pMP81 DNA recovered from the transformant was not rearranged at the gross sequence level as a result of transmission in U. maydis. Preliminary evidence suggests that the yeast 2-micron DNA promotes autonomous plasmid replication in U. maydis, albeit inefficiently, resulting in low transformation frequencies and plasmid copy numbers.

Abstract: Restriction endonuclease digestion of mitocondrial DNAs from the nine Dekkera/Brettanomyces yeasts have revealed that three separate pairs of species, namely D. bruxellensis/B. lambicus; B. abstinens/B. custersii and B. anomalus/B. clausenii have identical genomes. This observation suggests that such analysis of mtDNA could be an important procedure for yeast taxonomy. Sizes of mtDNAs showed a graded range from the 28 kbp molecule in B. custersianus to the 100 kbp molecule in B. custersii. Furthermore, although the mtDNAs from D. intermedia (72 kbp) and D. bruxellensis (82 kbp) differ in size by 10 kbp the restriction enzyme fragmentation patterns are generally similar. The differences are reminiscent of mtDNA polymorphisms found in strains of Saccharomyces cervisiae which result from insertions or deletions, chiefly within genic sequences. By analogy, the two Dekkera species may, on further analysis, be revealed as variants of a single species.

Abstract: We have examined the structure of the rRNA genes from the mitochondrial genome of Podospora anserina Using R-loop analysis, nuclease protection experiments, and Southern blot hybridization analysis we have observed two intervening sequences (IVS) in the large rRNA gene, and none in the small rRNA gene the IVS sequences are 165 kbp and 273 kbp long, and the larger of the two is in the position of the conserved IVS found in the mitochondrial genomes of other fungi We have detected precursor transcripts for the large rRNA, and these data support the observation of two IVS in this gene We also note that the large and small rRNA genes are separated by approximately 6 kbp of DNA

Abstract: Cloned chloroplast restriction endonuclease DNA fragments were used as hybridization probes to identify the in vivo transcriptional products of the chloroplast genome from the alga Euglena gracilis. Total cellular RNA was size fractionated by electrophoresis in denaturing gels and transferred to nitrocellulose paper. Individual plasmids containing specific chloroplast DNA fragments were radioactively labeled in vitro and hybridized to the immobilized RNA. The stable RNAs in the chloroplast were identified on the basis of their size and their origin on the chloroplast genome. Several transcripts were shown to be developmentally expressed. Some transcripts showed a possible precursor-product relationship. The rDNA was shown to be transcribed as a large transcript and then processed to the, mature rRNAs.

Abstract: Using the restriction endonucleases SaII, SmaI, BgII and KpnI, physical maps of chloroplast DNA isolated from normal and cytoplasmic male sterile (radish cytoplasm) lines of B. napus were constructed and compared. In this study, a rapid and simple procedure was developed for the isolation of chloroplast DNA restriction fragments from low gelling temperature agarose gels. The overall structural organization of N and cms B. napus appears to be rather similar to that of cpDNAs of other higher plants. It is composed of two identical sequences (each about 15 Md) arranged as an inverted repeat separated by two single copy-regions of different sizes (about 54 and 15 Md). In both genomes the ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat. The two chloroplast genomes are distinguished by a point mutation in the rRNA locus. Genes for the large subunit of ribulose-1.5-biphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by a minimum of 30 Md of DNA within the large single copy region.

Abstract: The photoreactivation repair gene (PHR1) of the yeast Saccharomyces cerevisiae was cloned in a hybrid plasmid (pJDB207), which is able to replicate as a multicopy episome in S. cerevisiae and Escherichia coli cells. The size of the DNA fragment found to have the photoreactivation activity was 3.0 kb, determined by recloning of the isolated fragment. In wild type cells transformed by the plasmid containing the PHR1 gene, the number of DNA photolyase molecules was 15 times greater than in wild type cells with pJDB207 only. Using the same receptor strain the excision repair gen RAD1 was also isolated. The size of the insert of the DNA which complements excision repair deficiency in recipient yeast cells was 5.7 kb. The recipient cells after transformation with the plasmid containing RAD1 showed the same UV-sensitivty as wild type cells with pJDB207 only.

Abstract: Two chromosomal mutations in yeast that result in oversecretion of the K1 killer toxin protein were examined. A recessive mutation in gene ski5 appears to lead to toxin oversecretion through a defect in a cell surface, PMSF-inhibited protease. A wild type killer strain degraded toxin following synthesis, and degradation could be partially prevented by addition of PMSF to the growth medium. The ski5 mutation caused an approximate ten fold oversecretion of toxin, similar to that seen in a PMSF-treated wild type culture, and no increased oversecretion in the presence of PMSF. The ski5 mutation caused oversecretion of other low molecular weight secreted proteins and appeared to oversecrete the α-factor pheromone, as judged by activity tests. The ski5 mutation was complemented by mutations in ski genes 1-4, and the mutant was not supersensitive to mating pheromones or K2 killer toxin.We also examined killer strains with a mutation in the nuclear gene krel which results in a defective (1→6)-β-D-glucan cell wall receptor for killer toxin. Such strains oversecrete toxin into the growth medium, but also, unexpectedly, oversecrete most other secreted proteins. The defect in (1→6)-β-D-glucan in these mutants appears to perturb the partitioning of secreted proteins between the cell wall and the medium.

Abstract: The three mutator strains ana r-8, ana r-14, and diu r-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions. Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe.

Abstract: Yeast strains carrying a functional MAL locus are inducible for the co-ordinate synthesis of both maltase and maltose permease when grown in the presence of maltose. Whether the maltose permease is encoded by a gene at the MAL loci has remained unclear due to the lack of mutants in this function. To isolate mutants defective in maltose transport, a positive selection strategy was employed in which a number of Mal− mutants were obtained. Among these one Mal- mutant was isolated which had normal levels of wild-type maltase in cell free extracts. This isolate, designated MGT1, has a defect in maltose transport (malT1), detected by its markedly lower uptake of [14C]maltose, and by its growth on media containing 10% but not 2% maltose. Since the Km of maltose uptake is altered 10-fold in this mutant and the Vmax remains unchanged, it is suggested that the mutation alters the structure of the maltose permease involved in transport of the disaccharide into the cell rather than its regulation.

Abstract: Chloroplast DNA isolated from a green alga Chlorella was shown by agarose gel electrophoresis and electron microscopy to contain a pair of large inverted repeat sequences of ca. 23 kbp. Electron microscopy revealed that the repeats were separated from each other by a small single strand loop of 29.5 kbp and a large single strand region of 98.5 kbp. Digestion with the restriction endonucleases Kpnl, Sstl, and Xhol, and hybridization with 32P-labelled tobacco rDNAs revealed that the genes for 16S and 23S rRNAs are present in the repeated sequences. From the hybridization pattern, a restriction map around the sequences were constructed, and the rRNA genes were found to be on the 10.8 kbp SstI fragment. This location was supported by electron microscopy (R-loop formation). Unlike Chlamydomonas reinhardii, Chlorella lacks a large intron in its 23S rRNA gene, and the 16S-23S spacer region is considerably long; the organization of rRNA operon is similar to that of higher plants.

Abstract: A hybrid plasmid (pLG4) containing pBR325 and the yeast arg4 gene was constructed then used to isolate DNA fragments of Chlamydomonas able to promote high frequency transformation of yeast. Three plasmids containing EcoRI restriction fragments of chloroplast DNA and two plasmids containing Aval fragments of nuclear DNA were shown to support autonomous replication of plasmids in yeast. The three EcoRI fragments correspond to restriction fragments R4, R5 and R11 of native chloroplast DNA. These fragments are clustered in the physical map of chloroplast DNA constructed by Rochaix (1978). All isolated plasmids were shown to transform yeast at high frequency but the yeast transformants were quite unstable mitotically. Potential cloning sites are still available in the new plasmids which could be used as vectors in yeast and possibly in Chlamydomonas itself.

Abstract: We have sequenced the gene for cytochrome c oxidase subunit 1 (CO I) in Neurospora crassa mitochondrial DNA. The gene is coded by the same strand as the rRNA and tRNA genes. The coding sequence predicts a protein of 557 amino acids, starting with methionine, and ending with asparagine. Comparison to the N-terminal amino acid sequence of the mature protein (Werner et al. 1980) reveals that the methionine is located at position -2. No other upstream AUG codons have been found in frame. The C-terminal part of the gene is about 70 basepairs longer than the corresponding parts of the Saccharomyces and mammalian genes. The homology between the Neurospora coding sequence and those of yeast and mammals is very high. As compared to Saccharomyces, the introns il through i5 are absent.

Abstract: We have previously demonstrated that the loss of Rcp-CEN3, a centromeric plasmid containing yeast rDNA autonomously replicating sequences (ARS) is as high as around 50% per generation for most yeast strains. In this study we have attempted to elucidate mechanisms underlying the high mitotic instability of Rcp-CEN3. For this purpose a tandem duplication of a rDNA ARS was constructed in Rcp-CEN3. The new plasmid having two ARSs possesses a markedly higher mitotic stability as compared to a monoARS Rcp-CEN3. The mitotic stability of this centromere-containing plasmid which has two replicators corresponds to the calculated value for the mitotic stability of two monoARS plasmids Rcp-CEN3 in given cells. Genetic analysis has demonstrated that both plasmids having one or two ARSs are maintained in the single copy state. These results demonstrate that the mitotic instability of centromeric plasmid Rcp-CEN3 carrying a rDNA ARS is associated with the absence of stringent control of replication from the rDNA ARS. A possible mechanism of replication of the chromosomal rDNA repeats in yeast is discussed in the light of this data.

Abstract: A diploid yeast thymidylate auxotroph was grown under conditions of thymidylate stress ranging from depletion to excess levels of the nucleotide. High concentrations of thymidylate were mutagenic and recombinagenic whereas starvation for thymine nucleotides was recombinagenic and only slightly mutagenic. These results are discussed in relation to possible mutagenic and recombinagenic mechanisms of nucleotide pool imbalances.

Abstract: HEM1, the structural gene for δ-aminolevulinic acid synthase, has been isolated on recombinant plasmids. A yeast genomic pool constructed in the E. coli — yeast shuttle vector YEp13 was used to clone the HEM1 gene by complementation. A leu2 hem1 yeast mutants was transformed with the yeast genomic pool and hybrid YEp13 plasmids carrying the HEM1 gene were cloned by their ability to complement both the leu2 and hem1 mutations in the recipient strain. The yeast transformants, bearing the HEM1-containing plasmids pYe(HEM1), showed a 24–28 fold increase in δ-aminolevulinic acid synthase activity and in the intracellular content of δ-aminolevulinic acid (5–8 fold) as compared to wild type strains, suggesting that the p(HEM1) gene is being expressed as a catalytically active enzyme which can be transported into the mitochondria. However, the transformant strains did not present higher-than-normal content of heme or cytochromes either in glucose or in glycerol media, indicating that the production of δ-aminolevulinic acid is not the rate-limiting step in heme biosynthesis in yeast.

Abstract: A complete clone bank representing the chloroplast DNA from Vicia faba has been constructed. A total of 15 fragments (10 Pst1, 1 Pst1-EcoR1 and 4 Sal1 fragments) were inserted into the vector pBR322 and transformed into the E. coli strain HB101. The cloned fragments were used as the main tools in constructing the physical map of Vicia faba for the restriction endonucleases Pst1, Kpn1 and Xho1. The identity of the cloned fragments was demonstrated by restriction analysis and blot hybridization. The information generated was used to construct the map. The 16S and 23S rRNA genes and the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase have been positioned on the map using heterologous probes. The orientation of the gene for the large subunit of RuBP carboxylase has also been determined.

Abstract: The cyanelle DNA from two different strains of Cyanophora paradoxa (strain LB555UTEX and strain 1555) was investigated. The cyanelle DNA from both strains showed a buoyant density in neutral CsCI gradients of 1.692 g/cm3. The total molecular weight, as judged by restriction endonuclease analysis, of the two cyanelle DNAs differed. In strain LB555UTEX the size of the cyanelle DNA was equivalent to 127 ± 1 kb whereas in strain 1555 a size of 138 ± 1 kb was consistently found. The sizes of individual DNA fragments and the number of recognition sites for a particular restriction endonuclease appeared largely unrelated. A high amount of cross hybridization, as judged by reciprocal heterologous DNA hybridizations, however indicated a high degree of sequence homology between the two cyanelle DNAs. Under comparable conditions, cyanelle DNA hybridized nearly exclusively with the dG+dC-rich rRNA transcription units from plastid DNAs. Up to now conserved restriction endonuclease recognition sites between the two cyanelle DNAs were only observed within the cyanelle rRNA genes which are present twice on both cyanelle DNAs.

Abstract: Any one of the inverted sequences present on the 2-µm DNA from Saccharomyces cerevisiae can promote replication of chimeric plasmids in Schizosaccharomyces pombe. When however a complete 2-µm molecule is present on the transforming plasmids, these are very unstable and systematically rearranged in S. pombe. Two types of transformation are observed in this case. One results from chromosomal integration of the incoming DNA. The second is dependent on a site specific recombination event between two molecules of the incoming DNA and results in a stably replicating dimeric structure. The choice between both pathways seems to depend on the expression of 2-µm function(s) in S. pombe.