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.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.

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.

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.