Genome sizes of 227 accessions of Gagea (Liliaceae) discriminate between the species from the Netherlands and reveal new ploidies in Gagea.

Nuclear genome size, as measured by flow cytometry with propidium iodide, was used to investigate the relationships within the genus Gagea (Liliaceae), mainly from the Netherlands. The basic chromosome number for Gagea is x = 12. The inferred ploidy in the Dutch and German accessions varies from diploid to decaploid. Consequently there is a large range of genome sizes (DNA 2C-values) from 14.9 to 75.1 pg. Genome sizes are evaluated here in combination with the results of morphological observations. Five species and the hybrid G. × megapolitana are reported. Apart from 14 diploid G. villosa, six plants of G. villosa with an inferred tetraploidy were found. For the 186 Dutch accessions investigated 85 turned out to be the largely sterile G. pratensis (inferred to be pentaploid). Inferred tetraploid and hexaploid G. pratensis were found in 30 and 20 localities, respectively. In one locality an inferred decaploid (10×) plant was found that could represent a doubled pentaploid G. pratensis. An inferred decaploid G. pratensis was never reported before. The genome size of Gagea × megapolitana from Germany fitted with its origin as a cross between the two hexaploids G. pratensis and G. lutea. Gagea spathacea from the Netherlands was inferred to be nonaploid as was recorded from plants across Europe. The aim of the study was to use flow cytometry as a tool to elucidate the taxonomic position of the Dutch Gagea.

Flow cytometric analysis of somaclonal variation in lineages of Hosta sports detects polyploidy and aneuploidy chimeras.

Somaclonal variation of some 124 specially selected cultivars of Hosta Tratt. (Hostaceae) was investigated. Nuclear DNA contents (2C-value) were measured by flow cytometry of leaves and roots of L1, L2 and L3 layers derived from apical meristems. These values were then converted to inferred ploidies by comparing the measured 2C-values and ploidy with those of the parent plant. During tissue-culture propagation, on occasion diploid (L1-L2-L3 = 2-2-2) hostas give rise to polyploids, such as fully tetraploids (4-4-4), and periclinal chimeras, such as partial tetraploids (4-2-2). Continual propagation can result in partial tetraploids becoming full tetraploids. Nuclear DNA of some diploids increased with incomplete chromosome sets resulting in fully aneuploids, such as hostas with a DNA ploidy of L1-L2-L3 = 2.5-2.5-2.5 and 3.7-3.7-3.7, and even in aneuploid periclinal chimeras, such as L1-L2-L3 = 2.5-2-2 and 3.8-2-2. The polyploidy of L1, irrespective of the ploidy of L2 and L3, is found to mainly determine the thickness of leaves. Also the higher the ploidy of L1, the wider and more intense in color is the leaf margin. The measurements of Hosta cultivars and their lineages of sports show that chromosome losses or gains are an important source of new cultivars. The complexity of chromosomal distribution in lineages of several Hosta cultivars is discussed.

Genome sizes for all genera of Cycadales.

Nuclear DNA content (2C) is reported for all genera of the Cycadales, using flow cytometry with propidium iodide. Nuclear DNA content ranges from 24 to 64 pg in cycads. This implies that the largest genome contains roughly 40 × 10(9) more base pairs than the smallest genome. The narrow range in nuclear DNA content within a genus is remarkable for such an old group. Furthermore, 42 of the 58 plants measured, covering five genera, have 18 chromosomes. They vary from 36.1 to 64.7 pg, covering the whole range of genome sizes (excluding the genome of Cycas). Hence, their does not seem to be a correlation between genome size and the number of chromosomes.

First nuclear DNA amounts in more than 300 angiosperms.

BACKGROUND AND AIMS: Genome size (DNA C-value) data are key biodiversity characters of fundamental significance used in a wide variety of biological fields. Since 1976, Bennett and colleagues have made scattered published and unpublished genome size data more widely accessible by assembling them into user-friendly compilations. Initially these were published as hard copy lists, but since 1997 they have also been made available electronically (see the Plant DNA C-values database http://www.kew.org/cval/homepage.html). Nevertheless, at the Second Plant Genome Size Meeting in 2003, Bennett noted that as many as 1000 DNA C-value estimates were still unpublished and hence unavailable. Scientists were strongly encouraged to communicate such unpublished data. The present work combines the databasing experience of the Kew-based authors with the unpublished C-values produced by Zonneveld to make a large body of valuable genome size data available to the scientific community. METHODS: C-values for angiosperm species, selected primarily for their horticultural interest, were estimated by flow cytometry using the fluorochrome propidium iodide. The data were compiled into a table whose form is similar to previously published lists of DNA amounts by Bennett and colleagues. KEY RESULTS AND CONCLUSIONS: The present work contains C-values for 411 taxa including first values for 308 species not listed previously by Bennett and colleagues. Based on a recent estimate of the global published output of angiosperm DNA C-value data (i.e. 200 first C-value estimates per annum) the present work equals 1.5 years of average global published output; and constitutes over 12 % of the latest 5-year global target set by the Second Plant Genome Size Workshop (see http://www.kew.org/cval/workshopreport.html). Hopefully, the present example will encourage others to unveil further valuable data which otherwise may lie forever unpublished and unavailable for comparative analyses.

Isolation and partial characterization of the Kluyveromyces lactis homologue of SKP1.

The SKP1 gene of Kluyveromyces lactis was isolated as a suppressor of a lethal temperature-sensitive mutation in the Saccharomyces cerevisiae CTF13 gene (Chromosome Transmission Factor 13). KlSKP1 was localized at chromosome V, adjacent to KlPAS3. A similar arrangement of the two genes is present in S. cerevisiae. Disruption of the KISKP1 gene was lethal, whereas overexpression of KlSKP1 lead to a decreased growth rate, to swollen and chain-forming cells with an increased DNA content, and to decreased plasmid stability. In both yeasts, promoter constructs lacking most of the purported binding sequence showed increased transcription levels of KlSKP1 in comparison to constructs with the entire promoter.

Isolation and characterization of KIUBP2, a ubiquitin hydrolase gene of Kluyveromyces lactis that can suppress a ts-mutation in CBF2, a gene encoding a centromeric protein of Saccharomyces cerevisiae.

The Kluyveromyces lactis UBP2 gene was isolated as a suppressor of a temperature-sensitive mutation in CBF2, a gene coding for a centromere-binding protein of Saccharomyces cerevisiae. The UBP genes are hydrolases than can cleave a ubiquitin moiety from a protein substrate. KlUBP2 is not essential for growth since a disruption of the KlUBP2 gene had little effect, except for a slight decrease in the growth rate. The stability of centromere-containing plasmids was not influenced either. In addition to KlUBP2, five S. cerevisiae genes involved in the ubiquitination pathway could suppress the ts-mutation in the CBF2 gene, namely UBA1, UBA2, UBP1, UBP2 and YUH1, although YUH1 was the only one that could do this like KlUBP2 from a single-copy plasmid. Surprisingly, these genes encode proteins with antagonistic activity as two, UBA1 and UBA2, are ubiquitin-activating enzymes whereas the other three are de-ubiquitinating hydrolases.

Characterization of the histidine mutants of Kluyveromyces lactis.

Thirty-eight different histidine mutations of Kluyveromyces lactis were isolated and genetically characterized. All of the mutations were nuclear recessive alleles. They turned out to belong to seven different complementation groups, designated hisA1 to hisA7. Five of these genes have been cloned by in vivo complementation of the Klhis mutations. Their homology to some of the histidine genes of Saccharomyces cerevisiae was confirmed by heterologous complementation. However, one of these KlHIS genes did not complement any mutation in the seven known histidine biosynthetic enzymes encoding genes (his1-his7) of S. cerevisiae.

The lysine-rich C-terminal repeats of the centromere-binding factor 5 (Cbf5) of Kluyveromyces lactis are not essential for function.

The gene coding for the centromere-binding factor 5 (CBF5) of Kluyveromyces lactis has been isolated by hybridization of a Saccharomyces cerevisiae CBF5 DNA probe to a K. lactis library. The amino acid sequence of KlCbf5 is highly homologous, 88% identity, to ScCbf5, but also to the rat protein Nap57 (64% identity). The main difference between both yeast proteins and the rat protein is the presence of a lysine-rich domain with KKE/D repeats in the C-terminal part of the protein. These repeats are thought to be involved in binding of the protein to microtubules. Deletion of the KKE/D domain in KlCbf5 however, has no discernible effect on growth on rich medium, sensitivity to the microtubule-destabilizing drug benomyl or segregation of a reporter plasmid. On the other hand, insertion of two leucine residues adjacent to the KKE domain increases the loss rate of a reporter plasmid. In both yeasts complementation of a lethal CBF5 disruption with the heterologous gene results in a slight increase in benomyl sensitivity. A possible role of CBF5 in chromosome segregation will be discussed.

Isolation and molecular analysis of the gene for cytochrome c1 from Kluyveromyces lactis.

By ethyl methanesulphonate mutagenesis of the yeast Kluyveromyces lactis we have isolated five nuclear mutants that were unable to grow on non-fermentable carbon sources. The mutations were found to belong to three complementation groups. After functional complementation of the mutation in one of these mutants we have cloned the structural gene for cytochrome c1, named KlCYT1. This gene has been assigned to chromosome VI and its nucleotide sequence exhibited 74.3% identity to the homologous gene of S. cerevisiae.

The red ade mutants of Kluyveromyces lactis and their classification by complementation with cloned ADE1 or ADE2 genes from Saccharomyces cerevisiae.

Seventy-six red adenine mutants of Kluyveromyces lactis were isolated. By complementation they could be assigned to two groups with 31 and 45 mutants. Transformation of several strains from each group with plasmids containing the Saccharomyces cerevisiae ADE1 or ADE2 gene showed that the largest group was ade2 and the other group was ade1. Several previously isolated 'ade1' mutants were classified to either group and given new gene and allele numbers. ADE1 was localized at chromosome III, closely linked to the mating type gene, making it a convenient marker for mating type. ADE2 was localized at chromosome V.

Centromere promoter factors (CPF1) of the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are functionally exchangeable, despite low overall homology.

The KlCPF1 gene, coding for the centromere and promoter factor CPF1 from Kluyveromyces lactis, has been cloned by functional complementation of the methionine auxotrophic phenotype of a Saccharomyces cerevisiae mutant lacking ScCPF1. The amino-acid sequences of both CPF1 proteins show a relatively-low overall identity (31%), but a highly-homologous C-terminal domain (86%). This region constitutes the DNA-binding domain with basic-helix-loop-helix and leucine-zipper motifs, features common to the myc-related transcription factor family. The N-terminal two-thirds of the CPF1 proteins show no significant similarity, although the presence of acidic regions is a shared feature. In KlCPF1, the acidic region is a prominent stretch of approximately 40 consecutive aspartate and glutamate residues, suggesting that this part might be involved in transcriptional activation. In-vitro mobility-shift experiments were used to establish that both CPF1 proteins bind to the consensus binding site RTCACRTG (CDEI element). In contrast to S. cerevisiae, CPF1 gene-disruption is lethal in K. lactis. The homologous CPF1 genes were transformed to both S. cerevisiae and K. lactis cpf1-null strains. Indistinguishable phenotypes were observed, indicating that, not withstanding the long nonconserved N-terminal region, the proteins are sufficiently homologous to overcome the phenotypes associated with cpf1 gene-disruption.

Mutational analysis of centromeric DNA elements of Kluyveromyces lactis and their role in determining the species specificity of the highly homologous centromeres from K. lactis and Saccharomyces cerevisiae.

The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KlCDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KlCDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20-80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of KlCDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level than a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.

Chromatin structures of Kluyveromyces lactis centromeres in K. lactis and Saccharomyces cerevisiae.

We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.

The consensus sequence of Kluyveromyces lactis centromeres shows homology to functional centromeric DNA from Saccharomyces cerevisiae.

The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5'-ATCACGTGA-3') flanked by an AT-rich (+/- 90%) stretch of +/- 160 bp followed by another conserved box (5'-TNNTTTATGTTTCCGAAAATTAATAT-3'). These three elements were named KlCDEI, KlCDEII, and KlCDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 bp AT-rich (+/- 90%) element, named KlCDE0, was found +/- 150 bp upstream of KlCDEI. The sequences of both KlCDEI and KlCDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161-164 bp in K. lactis versus 78-86 bp in S. cerevisiae and the presence in K. lactis of KlCDE0, which is not found in S. cerevisiae.

Centromeric DNA of Kluyveromyces lactis.

A direct selection method was used to isolate centromeres from a genomic library of the yeast Kluyveromyces lactis. The method is based on the lethality at high copy number of the ochre-suppressing tRNA gene SUP11. Five different chromosomal fragments were found that confer mitotic stability to plasmids containing a replication origin of K. lactis (KARS). In addition, KARS plasmids containing these fragments have a copy number of approximately one, and each of the five fragments hybridizes to a different chromosome of K. lactis. From these results we conclude that five of the six centromeres of K. lactis have been isolated. These centromeres do not function in S. cerevisiae.

The effect of glucose and manganese on adenosine-3',5'-monophosphate levels during growth and differentiation of Aspergillus nidulans.

The role of cyclic adenosine monophosphate (cAMP) during growth and development of Aspergillus nidulans was investigated. In normal cultures the highest amount of cAMP, expressed on a dry weight basis, was found after 24 h of growth when still more than 5% glucose was present in the medium. After depletion of the medium even a slight fall in cAMP was noted. Glucose concentrations ranging from 0.5-12% resulted in a slight decrease in the amount of cAMP as measured after 24 h of growth. Cultures with manganese deficiency resulted in a low cAMP level after 24 h of growth. However, the exhaustion of glucose in the absence of manganese was connected with a sharp increase in cAMP. This indicates that manganese shortage was not a direct cause of the low cAMP level after 24 h. The amount of cAMP rose with increasing concentration of manganese in the medium until a maximum at 0.25 muM. It is tempting to speculate that this rise in cAMP in the manganese deficient culture is explained by the absence of glucose, that in the control culture is derived from the breakdown of the reserve material alpha-1,3-glucan. Addition of manganese after glucose exhaustion to a manganese deficient culture induced cleistothecium formation. However, they contained only a few ascospores indicating the importance of alpha-1,3-glucan as a carbon and energy source for ascospore formation. The regulation of the level of cAMP by the transport of glucose into the cell or its intracellular concentration is discussed.

Sexual differentiation in Aspergillus nidulans: the requirement for manganese and the correlation between phosphoglucomutase and the synthesis of reserve material.

Aspergillus nidulans was completely devoid of fruit bodies when grown on manganese deficient cultures. This result was shown earlier to be due to a lack of alpha-1,3 glucan in the cell wall. Several enzymes of carbon and nitrogen metabolism were investigated in an attempt to explain the absence of this reserve material. Synthesis of glucose-6-phosphate dehydrogenase, phosphoglucoisomerase and aldolase, were not strongly affected by manganese deficiency. However, phosphoglucomutase showed only 60% of the activity of the control cultures and it was argued that this was connected with the low amounts of alpha-1,3 glucan synthesized. Malate dehydrogenase was the enzyme the least affected by manganese deficiency and the two to threefold higher activity measured after glucose depletion might indicate the induction of the glyoxylate cycle. An impaired glutamine synthetase could explain the increase in activity observed for NAD-glutamine dehydrogenase.

Sexual differentiation in Aspergillus nidulans: the requirement for manganese and its effect on alpha-1,3 glucan synthesis and degradation.

Aspergillus nidulans was grown on media with added amounts of manganese ranging from 0--2.5 muM. Manganese deficiency prevented cleistothecium development, although good vegetative growth was retained. Subsequent analysis of the mycelium produced under Mn2+ deficient growth revealed that alpha-1,3 glucan, the man carbon and energy source for fructification, was virtually absent from the cell wall. Several enzymes related to cell wall composition were investigated. Beta-1,3 glucanase, and very remarkably, alpha-1,3 glucanase reached about the same activity on the Mn2+ deficient and sufficient media, but amylase and protease were about 60 and 75% lower respectively on the Mn2+ deficient media and the correlation of these findings is discussed.