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.

Using 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 bp 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 bp in the rDNA enhancer, and 25 bp 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 (Pol I) transcription at the main terminator T2.

Termination of transcription by yeast RNA polymerase I.

Analysis of the termination of transcription by yeast RNA polymerase I (Pol I) using in vitro run-on experiments in both isolated nuclei and permeabilized cells demonstrated that Pol I does not traverse the whole intergenic spacer separating consecutive 37S operons, but terminates transcription before reaching the 5S rRNA gene, that is within NTS 1. In order to discriminate between processing and termination at the 3'-end generating sites previously identified in vivo in NTS 1 (T1, T2 and T3), fragments containing these sites were inserted into the middle of the reporter DNA of an artificial rRNA minigene. RNA isolated from yeast cells transformed with these minigenes was analyzed for the presence of transcripts derived from sequences both up- and downstream of the insert by Northern blot hybridization, reverse transcription analysis and S1 nuclease mapping. In accordance with previously obtained results T1 (+15 to +50) was found to behave as a processing site. T2 (+210) however was concluded to be an efficient, genuine Pol I terminator. In addition to T2, two other terminators were identified in NTS 1: T3A (at +690) and T3B (at +950). Surprisingly, when the 3' terminal part of NTS 2 was tested for its capacity to generate 3'-ends, another terminator (Tp) was found to be present at a position 300 bp upstream of the transcription initiation site of the 37S-rRNA operon.

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.

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.

Isolation, characterization, and evolutionary aspects of a cDNA clone encoding multiple neuropeptides involved in the stereotyped egg-laying behavior of the freshwater snail Lymnaea stagnalis.

The cerebral neurosecretory caudodorsal cells (CDCs) of the freshwater pulmonate snail Lymnaea stagnalis control egg laying, an event that involves a pattern of stereotyped behaviors. The CDCs synthesize and release multiple peptides, among which is the ovulation hormone (CDCH). It is thought that each peptide controls a specific aspect of the processes involved in egg laying. We isolated and characterized a CDC-specific cDNA clone that encodes the ovulation hormone (CDCH). RNA blot analysis and in situ hybridization experiments demonstrated that the CDCs are the major cell groups in the cerebral ganglia that transcribe the CDCH gene. In addition to CDCH, the 259-amino acid-long CDCH preprohormone contains 11 other predicted peptides. The overall homology of the CDCH preprohormone with the egg-laying hormone (ELH) preprohormones of the marine opisthobranch snails Aplysia californica and A. parvula is very low (29 and 26%, respectively). However, a more detailed comparison revealed a highly differential pattern of conservation of peptide regions. Significant homology was found between the regions containing (1) CDCH and ELH, (2) repeated pentapeptides, (3) alpha-caudodorsal cell peptide and alpha-bag cell peptide, and (4) 2 regions representing as yet unidentified peptides. Insignificant homology was found when comparing regions containing the other predicted peptides. The conserved peptides probably control similar aspects of the egg-laying fixed action patterns in these distantly related gastropod species. The pentapeptide region exhibits the highest level of homology (75%); in addition, an extra pentapeptide has been generated on the CDCH precursor. This indicates a vital function of these peptides in Aplysia, as well as in Lymnaea species.

Growth-controlling molluscan neurons produce the precursor of an insulin-related peptide.

Insulin and related peptides are key hormonal integrators of growth and metabolism in vertebrates. There is little biochemical evidence for insulin-related peptides in invertebrates, apart from insects for which definitive structural information on these peptides (prothoracicotropic hormone, PTTH) has recently been obtained. We report here the first complete complementary DNA-derived primary structure of a preproinsulin-related protein from identified neurons in an invertebrate, the mollusc Lymnaea stagnalis. We have demonstrated by in situ hybridization that transcription of the gene for this molluscan insulin-related peptide (MIP) occurs in the cerebral light-green cells, giant neuroendocrine cells involved in the control of growth, as well as in a pair of neuroendocrine cells called the canopy cells. The insulin-related peptide precursor has the same overall structure as its vertebrate counterparts. The discovery of insulin-related peptides in invertebrates substantiates the evidence for a widespread and early evolutionary origin of the insulin superfamily.

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.

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.

The RNA polymerase I initiation site and the external transcribed spacer of the fission yeast Schizosaccharomyces pombe ribosomal RNA genes.

A 5.45-kb fragment containing the 5' end of the ribosomal RNA transcriptional unit from the fission yeast Schizosaccharomyces pombe was cloned in the yeast-E. coli shuttle vector YEp13. The transcription start point was mapped by R looping and S1 nuclease protection. The sequence of the entire external transcribed spacer (ETS) and its flanking regions was determined. Comparison of the sequence around the transcription start point with those of four budding yeasts (Saccharomycetoideae) reveals a consensus sequence from position -9 to -4 from the start. This sequence is likely to be an important element of the promoter for yeast RNA polymerase I (Pol.I). Comparison of all known Pol.I promoter sequences reveals a strong bias for nucleotides (nt) at several positions between -16 and +10. These nt may have a critical role in the transcription initiation process. The S. pombe ETS, which comprises 1355 bp, is significantly longer than those of the budding yeasts and lacks any significant sequence homology with the Saccharomyces cerevisiae ETS. R-loop analysis reveals a putative processing site within the ETS of S. pombe.

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.

Deletion mapping of the yeast Pol I promoter.

Deletions in the promoter region of the 37S pre-rRNA operon in yeast were constructed and analysed in vivo using an artificial ribosomal minigene present on an extrachromosomal yeast vector. Sequences required for correct transcription initiation were found to be located between positions -192 and +15 relative to the start; a 5'-deletion down to position -133 reduces the transcription yield of the minigene at least five-fold. To allow detection of transcription of the minigene in isolated nuclei of yeast transformed with a minigene-bearing plasmid we attempted to increase the minigene copy number. The transcription yield in vivo appeared not to be proportional to the copy number but was found to be greatly enhanced when two or three minigenes are present in tandem. alpha-Amanitin sensitivity of transcription of these minigenes in isolated nuclei proved that RNA polymerase I is responsible for their transcription.

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.

Structure and function of the nontranscribed spacer regions of yeast rDNA.

The sequences of the nontranscribed spacers (NTS) of cloned ribosomal DNA (rDNA) units from both Saccharomyces cerevisiae and Saccharomyces carlsbergensis were determined. The NTS sequences of both species were found to be 93% homologous. The major disparities comprise different frequencies of reiteration of short tracts of six to sixteen basepairs. Most of these reiterations are found within the 1100 basepairs long NTS between the 3'-ends of 26S and 5S rRNA (NTS1). The NTS between the starts of 5S rRNA and 37S pre-rRNA (NTS2) comprises about 1250 basepairs. The first 800 basepairs of NTS NTS2 (adjacent to the 5S rRNA gene) are virtually identical in both strains whereas a variable region is present at about 250 basepairs upstream of the RNA polymerase A transcription start. In contrast to the situation in Drosophila and Xenopus no reiterations of the putative RNA polymerase A promoter are present within the yeast NTS. The strands of the yeast NTS reveal a remarkable bias of G and C-residues. Yeast rDNA was previously shown to contain a sequence capable of autonomous replication (ARS) (Szostak, J.W. and Wu, R (1979), Plasmid 2, 536-554). This ARS, which may correspond to a chromosomal origin of replication, was located on a fragment of 570 basepairs within NTS2.

The in vivo and in vitro initiation site for transcription of the rRNA operon of Saccharomyces carlsbergensis.

We have performed a detailed analysis of the transcription initiation of the rRNA operon in the yeast Saccharomyces carlsbergensis. Electron microscopic analysis of R-looped pre-rRNA molecules together with a very sensitive S1-nuclease mapping showed the use of only a single transcription start at about 700 bp upstream of the 17S rRNA gene and not of the minor start sites proposed for the very closely related species S. cerevisiae by others [Bayev et al. (5), Swanson and Holland (6)]. The sequence of 730 bp of the initiating region is presented. In vitro transcription in concentrated lysates of yeast spheroplasts in the presence of (gamma-SH)ATP or (gamma-SH)GTP, followed by purification of the in vitro initiated RNA via Hg-agarose, revealed that on the endogenous template exactly the same site is used for transcription initiation as in vivo.

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.

We 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 Saccharomycetoideae. 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.

A conserved sequence element is present around the transcription initiation site for RNA polymerase A in Saccharomycetoideae.

To identify DNA elements involved in the initiation of rRNA transcription in yeast we located the start site of the rRNA operon of Kluyveromyces lactis and Hansenula wingei, both members of the Saccharomycetoideae, by S1 nuclease analysis and determined the surrounding nucleotide sequences. Comparison of these sequences with those of Saccharomyces carlsbergensis, S. cerevisiae and S. rosei (all belonging to the same yeast subfamily) reveals an identical sequence at the site of transcription initiation from position +1 to +7 which is part of a larger conserved region extending from position -9 to +23; the conserved heptanucleotide sequence is supposed to constitute an important part of the promoter for yeast RNA polymerase A. The non-transcribed spacers (NTS) upstream of position -9 have diverged strongly with the exception of two short elements around positions -75 and -135. The external transcribed spacer (ETS) downstream of position +23 is largely conserved between K. lactis, S. rosei and S. carlsbergensis except for a divergent region around position +75. On the other hand, the ETS of H. wingei has diverged significantly.

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.

The primary and secondary structure of yeast 26S rRNA.

We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence. The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence. Starting from the recently proposed structure of E. coli 23S rRNA (see ref. 25) we constructed a secondary structure model for yeast 26S rRNA. This structure is composed of 7 domains closed by long-range base pairings as n the bacterial counterpart. Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes. The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II. Domain VI, which is extremely conserved, is probably part of the ribosomal A site. alpha-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI. Nearly all of the located methyl groups, like in E. coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure. The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in E. coli 23S rRNA to be at the interface of the ribosome.