STUDIES ON DIPLOID STRAINS OF DICTYOSTELIUM DISCOIDEUM

Ross, Ian K. (Yale U., New Haven, Conn.) Studies on diploid strains of Dictyostelium discoideum. Amer. Jour. Bot. 47 (1) : 54—59. Illus. 1960.–Three strains of Dictyostelium discoideum having the diploid number of chromosomes (14) at all stages of their life cycle were examined. No evidence of sexuality as shown by syngamy or meiosis was found in the diploid strains. Two of the diploid strains were unstable and reverted to the haploid type with 7 chromosomes. These haploid strains had a sexual phase in which syngamy and meiosis were observed. The nuclear behavior of both the diploid and haploid strains differed from that reported in previous papers.     

Chromosomal changes in eight cell strains of the tasmanian rat-kangaroo,Potorous tridactylis (Marsupialia). Male and female heart fibroblasts cultured in vitro

Eight cell strains, derived from the hearts of a male and a female Motorous, were followed during their in vitro cultivation.All three male cell strains started as normal diploid cell strains. One of them, 2Hpo stayed diploid until passage 59, when the cells were frozen and stored at –96°C. After a period of growth retardation, that lasted two months, 1Hpo showed aneuploidy, the cells having 22–24 chromosomes. The cells with 23 chromosomes formed about 30% of the population. These cells predominantly missed the chromosomes 2, 3 and Y₁, from the tetraploid set. In the other cells no consistent pattern was observed. The cell strain 4Hpo did not show aneuploidy after three months of growth retardation. At the last passage (nr. 24) before death, it showed 25% diploid cells and 40% tetraploid cells.Three female strains were initiated on fibrin clot, two on plasm clot. No differences in growth and chromosomal changes, due to the different embedding media, were observed. All the strains started as diploid (2n=12) cell strains. The chromosomal changes that occurred showed many differences. Three cell strains (5Hf, 7Hp, 52Hf) died without showing any pattern in the aneuploid cells. One cell strain (53Hf) showed an aneuploid cell population with a stemline of 14 chromosomes. The cell strain (8Hp) showed different changes in ploidy. After 50 passages, it changed from diploid to aneuploid (19 chromosomes per cell in the stemline). Twenty passages later diploid cells started to dominate the population again (80% at passage 85). Then a new aneuploid population with a stemline of 18 chromosomes (30% triploid cells) arose, and the strain survived as a permanent line.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract Nr. 052-64-I BIAN, and it also received support form the Foundation for Basic Medical Research (FUNGO).     

Chromosomal changes in three cell strains of the tasmanian rat-kangaroo,Potorous tridactylis,cultured in vitro (Marsupialia)

The chromosomal changes occurring in three strains of cells from Potorous tridactylis, one derived from testis and two of kidney tissue, were followed during the in vitro life of the strains.One kidney cell strain was a slow growing one and died after 23 passages showing aneuploidy with very aberrant metaphases. The strain derived from testis showed aneuploidy after a period of growth retardation, about 50% of the aneuploid cells having 18, 19 or 20 chromosomes. In these cells the chromosomes 1, 2 and 4 were always present in triplicate and the cells always had two X-chromosomes. The second kidney strain showed aneuploidy after a period of growth retardation, cells with 22 and 23 chromosomes being the most frequent ones, but in different proportions. As the number of aneuploid cells gradually decreased, diploid cells appeared in the population. Their number also decreased and a new population of aneuploid cells arose, having 23 chromosomes, missing one chromosome nr. 5 from the tetraploid complement. Then again the cell strain returned to diploidy but as the frequency of these diploid cells decreased, the strain died out.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract No. 052-64-I-BIAN, and it also received support from the Foundation for Basic Medical Research (FUNGO).     

Caffeine enhancement of radiation killing in different strains of Saccharomyces cerevisiae.

Summary Haploid and diploid wild type strains, and three classes of radiation-sensitive mutants of Saccharomyces cerevisiae were tested for enhancement of UV-inactivation by caffeine in growth medium. In addition, the sensitizing effect of caffeine was studied in a haploid and a diploid wild type strain after gamma-irradiation. The drug sensitized the UV-irradiated cells of all strains except those reported to be only slightly UV-sensitive but highly sensitive to ionizing radiation. After gamma-irradiation, no caffeine-enhancement of killing was observed in stationary phase cells of either the haploid or the diploid strain. However, log-phase cells of both strains were partially sensitized.The results of both sets of experiments suggested that caffeine interferes with a recombinational repair occurring in cells in S or G2 phase.     

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae

We describe a novel chromosome engineering technique for shuffling selected regions of chromosomes from two strains in Saccharomyces cerevisiae: The technique starts with the construction of MAT a and MATα strains in which a particular chromosome is split at exactly the same site in both strains such that the split chromosomes generated are marked with different markers. The two strains are then crossed, and the resultant diploid is cultivated in nutrient medium to induce loss of the split chromosome originating from either of the strains. We predicted that some of these clones that are hemizygous for the split chromosome would spontaneously restore a homozygous configuration of the split chromosome during cultivation. We verified this prediction by tetrad analysis and quantitative Southern hybridization analysis, indicating that it is possible to create diploid hybrids in which a selected region of a chromosome from one strain is replaced by the corresponding chromosomal region from another strain. We also found that some chromosomal segments maintain a hemizygous state. This novel technique, which we call ‘chromosome shuffling’, could provide a new tool to analyze phenotypic alterations caused by the replacement or hemizygosity of a selected chromosomal region in not only laboratory but also industrial strains of S. cerevisiae.     

The 5S ribosomal RNA gene clusters in Tetrahymena thermophila: strain differences, chromosomal localization, and loss during micronuclear ageing

Summary The organization of the 5S genes in the genome of Tetrahymena thermophila was examined in various strains, with germinal ageing, and the 5S gene clusters were mapped to the MIC chromosomes. When MIC or MAC DNA is cut with the restriction enzyme EcoRI, electrophoresed, blotted, and probed with a 5S rDNA probe, the banding patterns represent the clusters of the 5S rRNA genes as well as flanking regions. The use of long gels and 60 h of electrophoresis at 10 mA permitted resolution of some 30–35 5S gene clusters on fragments ranging in size from 30-2 kb (bottom of gel). The majority of the 5S gene clusters were found in both MIC and MAC genomes, a few being MIC limited and a few MAC limited. The relative copy number of 5S genes in each cluster was determined by integrating densitometric tracings made from autoradiograms. The total number of copies in the MAC was found to be 33% greater than in the MIC. When different inbred strains were examined, the majority of the 5S gene clusters were found to be conserved, with a few strain-specific clusters observed. Nine nullisomic strains missing both copies of one or more MIC chromosomes were used to map the 5S gene clusters. The clusters were distributed non-randomly to four of the five MIC chromosomes, with 17 of them localized to chromosome 1. A deletion map of chromosome 1 was constructed using various deletion strains. Some of these deletion strains included B strain clones which had been in continuous culture for 15 years. Losses of 5S gene clusters in these ageing MIC could be attributed to deletions of particular chromosomes. The chromosomal distribution of the 5S gene clusters in Tetrahymena is unlike that found for the well-studied eukaryotes, Drosophila and Xenopus.     

Geographical distribution and cytological variation ofLemna paucicostata Hegelm. in Japan

More than 100 strains ofLemna paucicostata Hegelm. collected from various localities in Japan were classified into 4 types according to the criterion of Yukawa and Takimoto (1976). N-1 type strains are widely distributed in Hokkaido, Honshu, Shikoku and Kyushu, N-2 type strains only in the area on the Japan Sea side of north Japan, K type strain only at the campus of Kyoto University, and S type strains in southern Japan. Numbers of chromosomes in somatic cells of N-1, N-2, K and S type strains were 2n=66–84, 40, 50 and 40, respectively. In N-2 and S type strains, all chromosomes (20 bivalent chromosomes) were lined up on the equatorial plate at metaphase of the first meiotic division (MI). However, in N-1 type strains, several chromosomes, and in the K type strain, more chromosomes were away from the equatorial plate at MI. Pollen fertility was 60–70%, higher than 90%, 0% and higher than 90%, in N-1, N-2, K and S type strains, respectively.     

Mating behavior and mate recognition pheromone blocking of male receptors in Brachionus plicatilis Müller (Rotifera)

Copulatory behavior of three S and three L type B. plicatilis strains from different geographic areas was analyzed. A 29 KD surface glycoprotein on females, characterized as a Mate Recognition Pheromone (MRP), binds to receptors in the corona of males and blocks mate recognition. Blocking was observed in all S and L strains even though the MRP was isolated from a single L-type strain. Binding was quantified using image analysis and a 20-fold difference was observed among strains. A direct relationship between the male discrimination of females and the intensity of MRP binding to male receptors was found. This relationship might be useful as a tool to examine variation in the mate recognition systems of other rotifer species.     

Chromosome polymorphisms close to the cm-ADE1 locus of Candida maltosa

The imperfect yeast Candida maltosa has an ill-defined genetic constitution; it is nominally diploid, but probably highly aneuploid, in nature. We report on polymorphisms specifically affecting those chromosomes which bear the cm-ADE1 gene. This gene encodes phosphoribosylaminoimidazole-succinocarboxamide synthetase, an enzyme in the adenine biosynthetic pathway. By electrophoretic karyotype analysis, three differently sized chromosomes were demonstrated to carry cm-ADE; the size (but not the number) of these chromosomes was also found to vary, both between strains and during the mitotic growth of a single strain. Four different alleles of cm-ADE1 have been cloned and sequenced from one prototrophic strain. DNA sequence divergence between these different alleles is as high as 8%, with the greatest divergence being found in the upstream region. Mitotic recombination events that led to changes in the karyotype were followed by using cm-ADE1 DNA as an hybridization probe. A recombination hot-spot in the neighbourhood of the gene appears to be responsible for the instability of the chromosomes on which it resides.     

A homozygous diploid subset of commercial wine yeast strains

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as ‘genome renewal’.     

Alternation of nuclear phase in the filamentous basidiomycete,Helicobasidium mompa

Variation in the number of nuclei and cellular ploidy were observed in eight strains ofHelicobasidium mompa. The basidiospores, single-spore isolates and field-isolated strains were all dikaryons. The cellular ploidy, which was assessed by analyzing the fluorescence emitted by DAPI-stained nuclei, was unstable: monokaryotic strains derived from the original dikaryotic strains by successive subcultures were mainly tetraploid, although the original dikaryon was in most cases diploid. On the other hand, a dikaryotic strain derived by treatment with benomyl was haploid. These results suggest that diploid dikaryon is a normal nuclear phase ofH. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.     

Chromosomal rearrangements during vegetative growth of a wild strain of Saccharomyces cerevisiae.

Electrophoretic karyotypes of two strains of Saccharomyces cerevisiae, a haploid laboratory strain and a wild strain known to be at least diploid, have been checked during vegetative growth. The karyotype of the haploid strain was very stable; however, the diploid strain underwent frequent modifications. In most cases the number of bands was reduced, but occasionally we observed one band splitting into two. In one case, chromosomal rearrangements took place between differently sized copies of chromosomes I and VI. We concluded that the chromosome length polymorphism observed among wild strains of S. cerevisiae could be explained partly by chromosomal structure reorganization occurring during mitosis.     

Use of disomic strains to study the arrangement of ribosomal cistrons in saccharomyces of ribosomal cistrons in Saccharomyces cerevisiae

Summary Disomic strains ofSaccharomyces cerevisiae were studied by DNA-rRNA hybridization to examine the arrangement of rRNA cistrons on yeast chromosomes as well as to identify a disomic strain which was enriched for rRNA cistrons. Four of the five disomic strains tested showed a per cent hybridization lower than wild type. Two of these strains were found to be disomic for more than one chromosome. A slight increase in the per cent hybridization was observed with DNA isolated from one disomic strain. It was concluded that some chromosomes inSaccharomyces cerevisiae had few if any rRNA cistrons suggesting that the rRNA cistrons are non randomly distributed over the genome. From DNA-tRNA hybridization experiments, evidence for the presence of tyrosine tRNA genes on chromosomes VI was obtained.     

A further study on chromosome minimization by protoplast fusion in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1–12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 μg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.     

Deacidification of grape juice with derepressed mutants of the yeast Hansenula anomala

Summary Transport and utilization of malic acid by the yeast Hansenula anomala are subject to glucose repression. Derepressed diploid mutant strains were obtained by hybridization of derepressed haploid mutant strains of opposite mating type. Six diploid mutant strains displayed derepressed behaviour with respect to malic acid utilization in the presence of glucose up to 30% (w/v). Three of these diploid mutant strains, as compared with the parent strain, were able to degrade completely malic acid in grape juice without fermenting the sugars. In addition, using one diploid mutant strain together with a strain of the wine yeast Saccharomyces cerevisiae, it was possible to carry out a mixedmicrovinification in which deacidification occurred simultaneously with alcoholic fermentation.     

Assignment of RFLP linkage groups to their respective chromosomes in aneuploids of sugi (Cryptomeria japonica)

Aneuploids of sugi (Cryptomeria japonica) were found in the open-pollinated progenies of triploidplus tree clones. Seven trisomics and one hypotriploid were used to assign the chromosomes to the RFLP linkage groups constructed previously. The Southern blots containing their genomic DNA were hybridized with the labeled DNA clones corresponding to the loci in the linkage map. The additional dosage in autoradiographs showed that the cloned DNA fragment was located on the extra chromosome in the trisomics. On the other hand, the extra chromosome in two trisomics and the chromosome lacking the triplet in the hypotriploid were cytologically identified as chromosome 10 by consistent presence of a secondary constriction in the proximal region of its short arm. As a result, three linkage groups were assigned to their respective chromosomes, namely chromosome 10 and two other chromosomes.     

Repair of DNA double-strand breaks requires two homologous DNA duplexes

DNA repair and cell survival in haploid and its diploid derivative strains ofSaccharomyces cerevisiae were studied after 100 krad X-ray irradiation. The cells were in theG ₁ stage of the cell cycle, where haploid cells had only one copy of genetic material per genome and diploid had two copies. It was found that diploid could repair double-strand breaks in its DNA after 48 hr of liquid holding which was accompanied by a four-fold rise in survival. In contrast a haploid strain failed to repair its DNA and showed no increase in survival after liquid holding. It is concluded that (1) repair of DNA double-strand breaks requires the availability of two homologous DNA duplexes, (2) restoration of cell viability during liquid holding is connected with repair of DNA double-strand breaks and (3) this repair is a slow process possibly associated with slow finding and conjugation of homologous chromosomes.     

Application of sequential integration for metabolic engineering of 1,2-propanediol production in yeast

The yeast species Saccharomyces cerevisiae was engineered to produce 1,2-propanediol (1,2-PD) using the delta/UB sequential gene integration method. To study the effects of increased copy number, 2 genes (mgs and gldA from Escherichia coli) were sequentially integrated into the chromosomes of S. cerevisiae strains of opposite mating type. The resulting strains (containing 0-3 copies of either mgs or gldA) were mated to create all possible combinations of the 2 genes introduced for 1,2-PD production. Enzyme activities were generally correlated with copy number, although there was greater variation in GldA activity in the diploid cells. The integrated genes were confirmed by Southern blot and 1,2-PD production was analyzed by HPLC. The strain containing 3 copies of mgs and gldA showed the highest level of 1,2-PD; however, 1,2-PD concentration was not clearly related to gene copy number. 1,2-PD production did correlate with Mgs specific activity, and high GldA specific activity was found to be inhibitory.     

A plant culture (BY‐2) widely used in molecular and cell studies is genetically unstable and highly heterogeneous

Nicotiana tabacum (tobacco, 2n = 4x = 48) is an allotetraploid with 24 S‐genome chromosomes (from a diploid related to N. sylvestris) and 24 T‐genome chromosomes (from a diploid related to N. tomentosiformis). The BY‐2 suspension cell culture, derived from N. tabacum cultivar Bright Yellow 2, has been used extensively for research in molecular and cell biology for almost 40 years; a Web of Knowledge search reveals that it has been used over 150 times since 2008 alone, largely for cell cycle and plant physiology studies. However, we show that this culture is unstable and, as with other long‐term cultures, exists as a community of cells with different karyotypes reflected in different chromosome numbers, morphologies and distributions of satellite repeats, At least one rearranged chromosome type was found in all cells investigated in detail. In comparison with N. tabacum, one satellite repeat, NTRS, has become dispersed across several chromosomes and there is complete homogenization of 35S rRNA genes towards T‐genome type rDNA units. Karyotype divergence should be considered when using BY‐2 cells for plant physiology or cell cycle/development studies in the future. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 459–471.     

THE KARYOTYPES OF DIPLOID CESPITOSE ZINNIAS: A METHOD AND ANALYSIS

The karyotypes of the three diploid (n = 10) species of the subg. Diplothrix (Zinnia—Compositae) were compared to determine whether there were any demonstrable differences which could then be sought in their polyploid derivatives. Because many of the chromosomes in a set were too similar to distinguish confidently between them, a method of analysis was developed which measures the similarity of whole sets of chromosomes rather than individual ones. The method consists of measuring the distances between graph-plotted vertices representing arm lengths of chromosomes of real or paper hybrids and then comparing these distances by means of U tests with those similarly derived for the “parents.” This procedure obviates the need of attempting to identify morphologues (morphologically similar chromosomes) in a somatic diploid root-tip cell and to equate corresponding pairs of chromosomes from different cells of a single plant or from different species or hybrids. No demonstrable differences in the karyotypes of diploid cespitose zinnias were found. Analysis of previously published data by this method indicated that there has been a general non-objectivity and non-operationalism in the determination of homologous chromosomes, and a general but unwarranted assumption that morphologues are in reality genologues (genetically corresponding chromosomes).