H. Yde Steensma

Four Arabidopsis thaliana 14-3-3 protein isoforms can complement the lethal yeast bmh1 bmh2 double disruption

The 14‐3‐3 proteins comprise a family of highly conserved proteins with multiple functions, most of which are related to signal transduction. Four isoforms from the plant Arabidopsis thaliana were able to complement the lethal disruption of the two Saccharomyces cerevisiae genes encoding 14‐3‐3 proteins; one complemented very poorly and one did not complement. However, the expression of the latter two isoforms was very low. These results show that at least four of the six A. thaliana isoforms are able to perform the same function(s) as the yeast 14‐3‐3 proteins.

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.

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.

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.