A system to study transcription by yeast RNA polymerase I within the chromosomal context: functional analysis of the ribosomal DNA enhancer and the RBP1/REB1 binding sites.

We have developed a novel system to study transcription by yeast RNA polymerase I (Pol I) of mutated rDNA units within the chromosomal context. For this, complete rDNA units carrying specific oligonucleotide tags in both the 17S and 26S rRNA genes were integrated into the chromosomal rDNA locus. Using this novel system, we analysed the action of the rDNA enhancer in stimulating transcription within the chromosomal context. We found that the enhancer acts as a stimulatory element in both directions, mainly on its two most proximal rRNA operons. Deletion of the sequences between the enhancer and the Pol I promoter in the tagged, integrated unit indicated that this part of the intergenic spacer contains no other transcriptional regulatory elements for Pol I. We also applied the system to study the function of the rDNA binding protein RBP1/REB1. For this purpose, we analysed tagged units in which either one or both of the binding sites for this protein have been inactivated. We found that mutations of both binding sites strongly diminish the transcription of the adjacent operon. The protein is hypothesized to play a crucial role in keeping the chromosomal rDNA units in an optimal spatial configuration by anchoring consecutive enhancers and promoters to the nucle(ol)ar matrix.

Linker scanning of the yeast RNA polymerase I promoter.

To define the RNA polymerase I promoter in the rDNA of Saccharomyces cerevisiae more precisely, we have constructed a series of 5'- and 3'-deletion mutants in a novel, plasmid-borne rDNA minigene, that also contains the transcriptional enhancer. Our data show that the Pol I promoter, in this context, extends from position -155 to +27, with 5'-deletions up to -134 and 3'-deletions up to -2 removing essential sequence information. To investigate the internal organization of the yeast Pol I promoter, linker scanning mutants were constructed, that traverse the Pol I promoter region and comprise between 5 and 12 clustered point mutations. Analysis of minigene transcription in yeast cells transformed with these plasmids demonstrates that the pol I promoter consists of three domains. Mutations in Domain I (from position -28 to +8) and Domain II (-70 to -51) drastically reduce promoter activity, whereas clustered point mutations in Domain III (starts at position -146 and presumably extends to position -76) appear to have less effect. Furthermore, the insertion of 4 nt between Domains I and II diminishes minigene transcription, indicating that the relative positions of these domains is essential.

[Ultrasonic examination in biliary tract diseases]. / U'ltrazvukovoe issledovanie pri zabolevaniiakh zhelchevyvodia- shchikh putei.

The authors consider that ultrasonic examination is a simple, safe and highly informative method of examination having no contraindications. Due to its high informative value the USE is of decisive significance in diagnosis of cholelithiasis. The obtaining of more exact diagnosis of "acalculous cholecystitis" requires strict interpretation of all the findings of ultrasonic examination. The ultrasonic symptomatology of noninflammatory diseases of the bile duct requires more exact details.

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.

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 ribosomal DNA in chloroplasts of Spirodela oligorhizaa.

The genes for the two large ribosomal RNAs (16S and 23S) and for the 4.5S rRNA in Spirodela oligorhiza chloroplast DNA are transcribed as one large, 7,000 nucleotides long precursor rRNA.Using S1-nuclease mapping, we have determined that the transcript ends 135 nucleotides 3' distal of the 4.5S rRNA gene. 5S rRNA therefore, is most likely transcribed separately.Northern blotting of chloroplast RNA with distinct probes derived from the rDNA region reveals RNAs, which can be described as intermediates in the processing of the large precursor. With these findings a pathway for the maturation of this precursor is proposed.

The nucleotide sequences of the regions flanking the genes coding for 23S, 16S and 4.5S ribosomal RNA on chloroplast DNA from Spirodela oligorhiza.

The nucleotide sequences of the flanking regions of the genes coding for Spirodela oligorhiza chloroplast ribosomal RNA's have been determined. We have compared these sequences to the corresponding ones in chloroplast DNA of other plants and of E. coli and find a striking sequential or structural homology. The region 5'-proximal to the gene coding for 16S rRNA contains a gene coding for tRNAval, which is transcribed from the same strand. In this area three prokaryotic promoter motifs are found: one located in front of the tRNAval gene and two in the intergenic space between this gene and the 16S rRNA gene. The middle one is used for the start of the transcription of the large ribosomal RNA precursor.

The nucleotide sequence of the 4.5S and 5S rRNA genes and flanking regions from Spirodela oligorhiza chloroplasts.

The base sequence of Spirodela oligorhiza chloroplast DNA coding for 4.5S and 5S ribosomal RNA, the flanking regions and the spacer between these two genes has been determined. We have compared these sequences with the corresponding ones in other higher plants. Besides a high degree of homology, some interesting differences are found.