B. J. M. Zonneveld

Nuclear DNA contents of all species of Helleborus (Ranunculaceae) discriminate between species and sectional divisions

Abstract. Genome size (C-values) and pollen viability staining were applied as new criteria to investigate the species of the genus Helleborus Linnaeus (Ranunculaceae). All species have the same chromosome number (2n=32). However, the nuclear DNA content, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 19 pg to 35.7 pg. The different genome sizes of the species coincided to a large extent with earlier determined section boundaries based on morphology. Flow cytometry can be a convenient method to discriminate between some species.

Centromeric DNA of Kluyveromyces lactis.

SummaryA 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 or 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 consensus sequence of Kluyveromyces lactis centromeres shows homology to functional centromeric DNA from Saccharomyces cerevisiae

SummaryThe 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 by followed by another conserved box (5′-TNNTTTATGTTTCCGAAAATTAATAT-3′).These three elements were named K1CDEI, K1CDEII, and K1CDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 by AT-rich (±90%) element, named K1CDE0, was found ± 150 by upstream of K1CDEI. The sequences of both K1CDEI and K1CDEIII 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 by in K. lactis versus 78–86 by in S. cerevisiae and the presence in K. lactis of K1CDEO, which is not found in S. cerevisiae.

Taxonomic implications of genome size and pollen colour and vitality for species of Agapanthus L’Héritier (Agapanthaceae)

Nuclear DNA content (2C) and pollen vitality and colour are used as new criteria to investigate all species of the genus Agapanthus L’Héritier. The species have the same chromosome number (2n=2x=30), with exception of four triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 22.1–31.6 pg. This implies that the largest genome contains roughly 1010 more base pairs than the smallest. The species could be divided in two groups based on pollen colour and DNA content: a group with lilac pollen and a DNA content between 22.3 and 24.1 pg containing the species A. campanulatus Leighton, A. caulescens Sprenger and the rarer A. coddii Leighton, and a group with yellow/brownish pollen and a DNA content from 25.2–31.6 pg containing the species A. praecox Willd., A. inapertus Beauv. and A. africanus (L.) Hoffmanns. Four other taxa, recognized by Leighton (1965) are treated as follows: A. comptonii Leighton, has a nuclear DNA content similar to A. praecox and is considered to be a synonym of A. praecox subsp. minimus Leighton.A. walshii L. Bol., has with 31.6 pg the same high amount of DNA as A. africanus from the same area and is therefore renamed as a subspecies (A. africanus subsp. walshii (Leighton) Zonn. & Duncan comb. nov.). The nuclear DNA amounts of A. dyeri Leighton, including the geographically isolated plants from Mozambique, are shown to be identical to A. inapertus. Therefore A. dyeri is considered synonymous with A. inapertus subsp. intermedius Leighton. A. nutans Leighton is identical in DNA content to A. caulescens and is considered to be synonymous with that species. Concluding there are six species: A. campanulatus Leighton, A. caulescens Sprenger, A. coddii Leighton, A. praecox Willd., A. inapertus Beauv. and A. africanus (L.) Hoffmanns. Nuclear DNA content as measured by flow cytometry and pollen colour are shown to be relevant traits to throw light on the relationships between Agapanthus species.

Mutational analysis of centromeric DNA elements of Klayveromyces 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 KICDEI, 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 KICDEI 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 K1CDEII 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 then 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.

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 non-conserved N-terminal region, the proteins are sufficiently homologous to overcome the phenotypes associated with cpf1 gene-disruption.

Flow cytometric analysis of DNA content in Hosta reveals ploidy chimeras

Nuclear DNA content in 84 Hosta cultivars was measuredby flow cytometry. As an internal standard we used the closely related Agave americana, the latter was calibrated against other reference species. Propidium iodide values were 20%higher than DAPI values, indicating a lower AT/GC ratio for Hosta. Ten new tetraploid plants were found in addition to the only tetraploid known so far; H. ventricosa.Small forms of the tetraploid H. ventricosa turned out tobe triploid hybrids. Three other triploids were discovered. Threesupposed tetraploids turned out to be diploid. In five plantsthe ploidy levels of the germ layers L1 and L3 appeared to be different. In these cases thick leaves coincides with a tetraploid DNA content. The DNA contents as measured in theseploidy chimeras coincide with differences in leaf colour, thusgiving an opportunity to analyze the build-up of Hosta plants.In general, analysis of the nuclear DNA content can be used as a simple means to increase the possibilities for creating new cultivars in this economically important perennial.

Genome size for the species of Nerine Herb. (Amaryllidaceae) and its evident correlation with growth cycle, leaf width and other morphological characters

Nuclear DNA content (2C) is used as a new criterion to investigate nearly all species of the genus Nerine Herb. The species have the same chromosome number (2n = 2x = 22), with the exception of three triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 18.0–35.3 pg. This implies that the largest genome contains roughly 2 × 1010 more base pairs than the smallest. The species, arranged according to increasing genome size, fell apart in three groups if growth cycle and leaf width were also considered. A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species, a broad-leaved winter growing group with four species has a DNA content from 25.3–26.2 pg and a broad-leafed summer growing group has a DNA content of 26.8–35.3 pg and contains six species. If the presence of filament appendages and hairiness of the pedicels were also considered, the thirteen evergreen species could be further divided into a group without filament appendages or hairy pedicels with a DNA content of 18.0–18.7 pg. A second group without filament appendages but with hairy pedicels had a DNA content of 19.7–22.3 pg. And a third group with both filament appendages and hairy pedicels had a DNA content of 22.0–24.6 pg. The exception is N. marincowitzii that, despite a low DNA content and narrow leaves is summer growing. The broad-leafed group is further characterised by the absence of filament appendages and the absence of strongly hairy pedicels. The exception here is N. pusilla that, despite a high DNA content, has narrow leaves and minutely hairy pedicels. Nuclear DNA content as measured by flow cytometry is shown to be relevant to throw new light on the relationships between Nerine species.

Taxonomic implications of genome size for all species of the genus Gasteria Duval (Aloaceae)

Abstract.Nuclear DNA content (2C) is used as a new criterion to investigate all species of the genus Gasteria Duval including the three recently described species Gasteria polita van Jaarsv., G. pendulifolia van Jaarsv. and G. glauca van Jaarsv.. The 122 accessions investigated have the same chromosome number (2n=2x=14), with exception of three tetraploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with Propidium Iodide, is demonstrated to range from 32.8–43.2 pg. This implies that the largest genome contains roughly 1010 more base pairs than the smallest. Based on DNA content the species could be divided in five groups: G. rawlinsonii Oberm. with 32.8 pg, 13 mostly inland species with 34.3–36.0 pg, five coastal species with 36.5–39.0 pg and Gasteria batesiana Rowley with 43.2 pg. The thirteen species with 34.3–36.0 pg could be divided further, in a group of eight species occupying mainly very restricted areas with 34.3–35.1 pg and a second group of five species with 35.2–36.0 pg mainly occupying large areas. These five groups did not coincide very well with the two sections and four series of Gasteria based on a cladistic analysis by van Jaarsveld et al. (1994). Based on its long leafy branches, location in the centre of Gasteria species distribution and its by far lowest DNA content, G. rawlinsonii might be the most primitive member of the genus. Nuclear DNA content as measured by flow cytometry is shown to be relevant to provide additional information on the relationships between Gasteria species.

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

Abstract  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.