Jacobus Klootwijk

High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with a high mitotic stability under non-selective conditions. Since high stability can best be assured by integration of the vector into chromosomal DNA we have set out to design a vector that is able to integrate into the yeast genome in a large number of copies. The rDNA locus appeared to be an attractive target for such multiple integration since it encompasses 100-200 tandemly repeated units. Plasmids containing several kb of rDNA for targeted homologous recombination, as well as the deficient LEU2-d selection marker were constructed and, after transformation into yeast, tested for both copy number and stability. One of these plasmids, designated pMIRY2 (for multiple integration into ribosomal DNA in yeast), was found to be present in 100-200 copies per cell by restriction analysis. The pMIRY2 transformants retained 80-100% of the plasmid copies over a period of 70 generations of growth in batch culture under non-selective conditions. To explore the potential of pMIRY2 as an expression vector we have inserted the homologous genes for phosphoglycerate kinase (PGK) and Mn2+-dependent superoxide dismutase (SOD) as well as the heterologous genes for thaumatin from Thaumatococcus danielli (under the GAPDH promoter), into this plasmid and analyzed the yield of the various proteins. Under optimized conditions the level of PGK in cells transformed with pMIRY2-PGK was about 50% of total soluble protein. The yield of thaumatin in the pMIRY2-thaumatin transformants exceeded by about a factor of 100 the level of thaumatin observed in transformants carrying only a single thaumatin gene integrated at the TRP1 locus in chromosome IV.

3'-End formation of transcripts from the yeast rRNA operon.

Deletion analysis of artificial rRNA minigenes transformed into Saccharomyces cerevisiae revealed that a 110 bp long fragment corresponding to positions ‐36 to +74 relative to the 3′‐end of the 26S rRNA gene, is both necessary and sufficient for obtaining transcripts whose 3′‐termini are identical to those of 26S and 37S (pre‐)rRNA. These termini are produced via processing of longer transcripts because in an rna 82.1 mutant the majority of the minigene transcripts extend further downstream. Since the rna 82.1 mutation inactivates an endonuclease involved in the 3′‐processing of 5S pre‐rRNA it is concluded that the maturation of 37S‐ and that of 5S pre‐rRNA requires a common factor. Comparison of the spacer sequences between Saccharomyces carlsbergensis, Saccharomyces rosei and Hansenula wingei revealed several conserved sequence blocks within the region between +10 and +55. These conserved sequence tracts, which are part of a longer region showing dyad symmetry, are supposed to be involved in the interaction with the processing component(s). Deletion of the sequences required for the formation of the 3′‐ends of 26S rRNA and 37S pre‐rRNA revealed a putative terminator for transcription by RNA polymerase I situated at position +210. This site maps within a DNA fragment that also contains the enhancing element for rDNA transcription by RNA polymerase I.

A system for the analysis of yeast ribosomal DNA mutations.

To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.

Molecular cloning of the rDNA of Saccharomyces rosei and comparison of its transcription initiation region with that of Saccharomyces carlsbergensis

We have cloned one complete repeating unit of rDNA from Saccharomyces rosei and determined its physical and genetic organization. Heteroduplex analysis of the rDNA units from S. rosei and S. carlsbergensis shows that the nontranscribed spacers are largely nonhomologous in sequence, whereas the transcribed regions are essentially homologous. We also determined the transcription initiation site for the 37S precursor RNA on S. rosei rDNA. Sequence comparison of the region surrounding the site of transcription initiation for the 37S RNA with the corresponding region of S. carlsbergensis revealed extensive homology from position -9 downstream into the external transcribed spacer. Very little homology was observed between position -9 and -55, but some homologous tracts are present upstream from position -55.

Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica; cloning and analysis of two size classes of repeats

Southern blotting of DNA from the ascomycetous yeast Yarrowia lipolytica revealed two major size classes of DNA units coding for rRNAs, which differ in length by about 1000 bp. We have cloned an rDNA unit of each size class. R-looping experiments revealed that the rRNA genes of both units are uninterrupted; subsequent heteroduplex analysis showed that the size difference both units is located within the nontranscribed spacer. Sequence analysis revealed that a major part of these spacers consists of a complex pattern of repetitions in periodicities of up to about 150 bp and that the difference between both rDNA units are located mainly in this repetitive region. Apart from different lengths of the repetitive regions, both rDNA units also reveal extended microheterogeneity within their homologous parts. Furthermore, no gene for 5S rRNA was observed in the spacer region. Therefore, the organization of the spacer of Yarrowia rDNA is clearly different from that of Saccharomyces cerevisiae.

Evolution of yeast ribosomal DNA: Molecular cloning of the rDNA units of Kluyveromyces lactis and Hansenula wingei and their comparison with the rDNA units of other Saccharomycetoideae

SummaryWe have studied the evolution of the yeast ribosomal DNA unit to search for regions outside the rRNA genes that exhibit evolutionary constraints and therefore might be involved in control of ribosome biosynthesis. We have cloned one complete rDNA unit of Kluyveromyces lactis and Hansenula wingei and established the physical and genetic organisation of both units. Both species belong to the subfamily of the Saccharomycetoidea. The lengths of the rDNA units of K. lactis and H. wingei are 8.6 and 11.1 kb respectively, and both comprise the 5S rRNA gene in addition to the large rRNA operon. Sequence conservation was monitored by restriction enzyme mapping as well as heteroduplex analysis of the two cloned rDNA units with S. carlsbergensis rDNA. These analyses showed that, phylogenetically, K. lactis is closer to S. carlsbergensis than H. wingei. The non-transcribed spacers (NTS) of both K. lactis and H. wingei have diverged completely from S. carlsbergensis; moreover in H. wingei the NTS are about double the length of these in the other two species. The transcribed spacers of both K. lactis and H. wingei contain conserved tracts. A homologous sequence of about 60 bp was found in the middle of the external transcribed spacer of H. wingei upon heteroduplexing with S. carlsbergensis rDNA, whereas the sequence at the transcription initiation site itself was insufficiently homologous to form a duplex. The sequence of the homologous region was determined both in H. wingei and K. lactis and compared with that of S. carlsbergensis. The function of this conserved element within the external transcribed spacer is discussed.

Heterogeneity in the ribosomal family of the yeast Yarrowia lipolytica: genomic organization and segregation studies

The cloned r-DNA units of Yarrowia lipolytica [Van Heerikhuizen et al., 39 (1985) 213-222] and their restriction fragments have been used to probe blots of genomic DNA of this yeast. Wild-type and laboratory strains were shown to contain two-to-five types of repeated units, each strain displaying a specific pattern. By comparing their restriction patterns, we could localize the differences between units within their spacer region. Tetrad analysis strongly suggested a clustered organization of each type of repeat as well as the occurrence of meiotic exchanges within the r-DNA family. Chromosome loss was induced by benomyl and allowed to map several r-DNA clusters on the same chromosome. All those results indicate that the Y. lipolytica r-DNA gene family is quite different from other yeasts.

Transcription of an artificial ribosomal RNA gene in yeast.

We constructed an artificial yeast rRNA gene and studied its transcription after introduction into a recipient yeast strain. The artificial gene comprised a fragment containing the sequence from position ‐207 to +128 relative to the site of initiation of Saccharomyces carlsbergensis 37S pre‐rRNA, followed by a marker fragment from Spirodela oligorhiza chloroplast DNA and finally a fragment containing the sequence from position ‐36 to +101 relative to the 3′ end of the 26S rRNA gene. The resulting construct was cloned into the yeast‐Escherichia coli shuttle vector pJDB207. Both Northern blot hybridization and R‐loop analysis of RNA from transformed Saccharomyces cerevisiae cells revealed a discrete transcript of the expected length. S1 nuclease mapping as well as primer extension analysis showed that the major proportion of the transcripts was initiated at exactly the same site as 37S pre‐rRNA. These results show that the respective rDNA fragments contain the information for correct initiation of transcription and formation of the 3′ end. A minor proportion of the transcripts was initiated at a number of sites between positions ‐1 and ‐100 upstream of the predominant start. The proportion and the pattern of these upstream starts is affected by the vector context of the artificial rRNA gene.

A yeast ribosomal DNA-binding protein that binds to the rDNA enhancer and also close to the site of Pol I transcription initiation is not important for enhancer functioning

SummaryUsing the gel retardation assay we have identified a protein that can specifically bind to a site within the enhancer of the 37S pre-ribosomal RNA operon in yeast, as well as to a site 210 by upstream of the site of transcription initiation of this operon. This protein (RBP1) has been partially purified by means of heparin-agarose chromatography and protects 20 by in the rDNA enhancer, and 25 by in the initiation region, against DNase I in an in vitro footprinting assay. In vivo footprinting studies using methylation of intact yeast cells with dimethylsulphate, indicate that the same binding sites are occupied in vivo as well. Deletions that abolish binding of RBP1 to the enhancer in vitro, as well as linker insertions into the RBP1 binding site in the initiation region that strongly diminish in vitro binding of RBP1, have no effect whatsoever on the enhancement of rDNA transcription in vivo. This was studied by deletion/mutation of the RBP1 binding site in vitro in an artificial ribosomal minigene and measuring the effect on the minigene transcription in vivo in yeast cells, transformed with the deleted/mutated minigenes. It can therefore be concluded that binding of RBP1 is not an important parameter in the functioning of the rDNA enhancer in yeast. Using the same minigene system we also show that RBP1 is not involved in termination of RNA polymerase I (PolI) transcription at the main terminator T2.

Consequences of phosphoglycerate kinase overproduction for the growth and physiology of Saccharomyces cerevisiae

SummaryThe physiological consequences of overproduction of the homologous glycolytic enzyme 3-phosphoglycerate kinase (PGK), integrated in 80 PGK1 gene copies in the genome of Saccharomyces cerevisiae are described. This multiple integration and the strong PGK overproduction (maximum 47% of the total soluble cell protein) do not affect the maximal specific growth rate, but cause 40% reduction of the molar growth yield, compared with that of the wild-type host. The extra energy that is needed for protein overproduction is mainly provided by extra fermentation (respirofermentative growth), but respiration is also elevated compared with the reference strains. The increase in the specific oxygen uptake rate indicates that the respiratory capacity of the yeasts is higher than that in the wild-type host, in which the limited capacity of respiration is generally supposed to be at its maximal level at the critical dilution rate, and is thus responsible for the switch to respirofermentative growth. In a medium PGK1 gene copy integrant (about 25 copies), overproduction of 10%–12% PGK has a stimulating effect on the growth yield and energy efficiency. In these cells the growth benefits of overproduction of the glycolytic enzyme are higher than the disadvantages of extra protein synthesis. The overproduction of PGK has also consequences for the glucose affinity of the yeasts: In the more overproducing strain the Ks is increased, compared to its reference strains. Elimination of strong overproducing cells from a glucose-limited chemostat culture is caused by two factors: (a) the excision of the PGK genes from the genome, which is of minor importance for wash-out, but the induction process for this overall decline of overproduction, and (b) the physiological selection process for less overproducing cells, caused by differences in affinity for glucose, most obvious at µ ≈ 1/2µmax. However in batch culture and in a chemostat at low specific growth rates, all the overproducing strains show high genetic stability and constantly provide high PGK quantities.