The extracellular acid protease gene of Yarrowia lipolytica: sequence and pH-regulated transcription.

The gene encoding an acid extracellular protease (AXP) from Yarrowia lipolytica (Candida olea) 148 was cloned and the complete nucleotide sequence was determined. The amino acid sequence deduced from the nucleotide sequence reveals that the mature AXP consists of 353 amino acids with an M, of 37427. The gene also encodes a putative 17 amino acid hydrophobic prepeptide and a 27 amino acid propeptide containing no potential N-glycosylation sites. The mature extracellular enzyme is produced by cleavage between Phe and Ala. AXP is a member of the aspartyl family of proteases. AXP shows homology to proteases of several fungal genera and to human progastricin. The coding sequence is preceded by a potential regulatory region of 1982 bp. Transcription of both AXP and alkaline extracellular protease genes of Y. lipolytica 148 is regulated by the pH of culture.

Activity of promoter mutants of the yeast ribosomal RNA gene with and without the enhancer.

The promoter and enhancer of the rRNA gene of Saccharomyces cerevisiae have been studied using a nuclease S1 protection assay to detect transcripts of an rRNA minigene in transformed yeast. Analysis of 5' deletion mutants showed that DNA between -163 bp and -155 bp was important for promoter activity and that some DNA between -155 bp and -145 bp was essential. The importance of DNA far upstream from the initiation site was confirmed by showing that minigene expression was much reduced by linker scanner mutations clustered around -148 bp, -133 bp and -100 bp, and was abolished by mutations clustered around -118 bp. The enhancer for rRNA biosynthesis increased transcription from all of the five mutated promoters that were tested. The magnitude of the enhancer effects on weakly active promoters was two- to three-fold less than on the wild-type promoter. Expression of a minor transcript in a 5' deletion to -10 bp was substantially reduced by a mutation which altered two base pairs in the core sequence of the promoter-proximal REB1 binding site.

The yeast rRNA gene enhancer does not function by recycling RNA polymerase I and cannot act as a UAS.

The mechanism of action of the yeast rRNA gene enhancer was investigated by measuring transcription of an rRNA minigene, cloned into a multicopy plasmid, in transformed yeast. Expression of the minigene was increased when the enhancer was cloned either upstream of or downstream from the minigene. When an enhancer was present both upstream and downstream of the minigene, the upstream element was functionally dominant. The upstream enhancer was active in this construct in the absence of detectable read-through by any RNA polymerase. In a construct containing tandem rRNA minigenes, an enhancer element located between the two promoters activated transcription from both independently. Therefore, the enhancer does not appear to activate transcription by recycling RNA polymerase I molecules to the promoter. The enhancer also failed to activate transcription from the intact promoter of the yeast CYC1 gene, and was unable to functionally substitute for the natural upstream activation sequences (UASs) of this gene. Therefore, the enhancer functions differently to UASs of RNA polymerase II genes, and is probably polymerase-specific.

Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite.

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.

Upstream activation of ribosomal RNA biosynthesis in Saccharomyces cerevisiae.

Yeast was transformed with eight recombinants that contained an rRNA minigene and upstream elements of rDNA in different orientations in the multi-copy yeast-Escherichia coli shuttle vector, pJDB207. The effect of these elements of upstream rDNA on the initiation of transcription of the minigene at the site for rRNA biosynthesis was determined by using an S1 nuclease mapping procedure to measure the abundance of the minigene transcript in RNA from the yeast transformants. Transcription of the minigene was enhanced 3-fold by DNA within a 2.2 kb element more than 1.5 kb upstream from the initiation site. Inversion of the 2.2 kb element decreased expression of the minigene by 40%. This 2.2 kb element contained approx. 500 bp from the 25S rRNA coding region at the 3' end of the preceding rRNA gene and 1 kb of adjacent nontranscribed spacer rDNA. The enhancing activity was independent of interference from readthrough that might have contributed to the 7-fold decrease in minigene expression caused by removing all rDNA upstream from -209 bp.

Transcription of a yeast ribosomal RNA minigene in Saccharomyces cerevisiae.

A transcription system using intact yeast has been developed for investigating which sequences are implicated in the initiation of transcription of yeast rRNA genes. The system employs an rRNA minigene that consists of the initiation and termination sites for rRNA biosynthesis separated by approx. 700 base pairs of vector DNA in the Escherichia coli-yeast shuttle vector, pJDB207. Two recombinants containing this minigene were constructed; one retained all of the nontranscribed spacer DNA upstream from the initiation site, the other retained 208 base pairs of this DNA. Transcripts of this structurally unique minigene in RNA from yeast transformed with these recombinants were readily detected by nuclease S1 mapping. These transcripts were initiated at the site used by the host rRNA genes, were approx. 3-fold more abundant in the recombinant retaining all of the nontranscribed spacer and were less abundant when the yeast was not growing.

The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A.

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.

Convolution analysis of transcription by yeast DNA-dependent ribonucleic acid polymerase A. A mathematical method for studying ribonucleic acid chain elongation.

The rate of initiation of RNA synthesis catalysed by yeast RNA polymerase A on native calf thymus DNA decayed exponentially with a half-life of about 4.3 min. The rate constant for initiation was unaffected by preincubating the enzyme with DNA, or by decreasing the concentration of GTP 4-fold. The rate of RNA synthesis was constant for 15--20 min and then decreased. Each enzyme molecule made no more than one RNA molecule. In this situation, initiation, elongation and total RNA synthesis are related by a convolution integral. Solution of the convolution integral revealed that the rate of elongation was apparently biphasic. Analysis of the size of the RNA product showed that this biphasic profile arose because most but not all of the enzyme stopped RNA synthesis soon after initiation.

Mechanism of action of a microsomal inhibitor of protein synthesis potentiated by oxidized glutathione.

Extracts of microsomal fractions cause an inhibition of protein synthesis that is most pronounced in the presence of 0.1mm-GSSG and 0.01mm-GTP, and is abolished by thiol or 0.4mm-GTP (Scornik et al., 1967). Fractionation of microsomal extracts showed that this inhibition of protein synthesis was caused by an enzyme, nucleoside diphosphate phosphohydrolase. Direct inhibition of protein synthesis on detergent-treated polyribosomes by 0.1mm-GSSG was observed under conditions of GTP limitation induced by omission of a GTP-regenerating system, or addition of a nucleoside triphosphate diphosphohydrolase. Thus GSSG potentiated the inhibition of protein synthesis caused by an enzyme that promoted removal of GTP. The inhibition was abolished by adding 4mm-2-mercaptoethanol or 0.4mm-GTP. Nucleoside diphosphate phosphohydrolase was thought also to act by promoting removal of GTP, thus causing an inhibition of protein synthesis that was potentiated by GSSG.

The number and location of genes for 5S ribonucleic acid within the genome of Drosophila melanogaster.

DNA was prepared from wild-type and two mutant stocks of Drosophila melanogaster that differed in their dosage of the nucleolar organizer region. The relative amounts of DNA from the nucleolar organizer region in these preparations of DNA were determined by hybridization with (3)H-labelled 28S rRNA. As expected, the amount of (3)H-labelled 28S rRNA that hybridized was directly related to the dosage of nucleolar organizer region. No positive correlation was observed between the amount of (3)H-labelled 5S RNA that hybridized and the dosage of nucleolar organizer region. Thus genes for 5S RNA are located primarily, if not exclusively, outside the nucleolar organizer region. The haploid genome of the wild-type D. melanogaster used in this work has 106 genes for 28S rRNA and 96-105 genes for 5S RNA.

The utilization of genes for ribosomal RNA, 5S RNA, and transfer RNA in liver cells of adult rats.

The rates of synthesis of ribosomes, 5S RNA, and tRNA necessary to maintain the steady-state concentrations of these entities in liver cytoplasm of adult rats were determined. On the average, each liver cell in the adult rat synthesizes 650 ribosomes, 650 molecules of 5S RNA, and 11,000 molecules of tRNA each minute. The numbers of genes per liver cell for rRNA, 5S RNA, and tRNA were 330, 1660, and 13,000, respectively, as determined by RNA: DNA hybridization experiments. Thus, on the average, individual genes for rRNA, tRNA, and 5S RNA are transcribed twice a minute, once a minute, and once every 2.5 minutes, respectively, in the adult rat liver.