Cloning and sequencing of the tuf genes of Streptomyces coelicolor A3(2).

Two tuf genes are present in Streptomyces coelicolor A3(2), which have been cloned and sequenced. These genes show a high degree of nucleotide sequence identity to the tuf1 and tuf3 genes of Streptomyces ramocissimus: the tuf1 genes are 94% identical, the tuf3 genes 87%. S. coelicolor tuf1 encodes a protein of 396 amino acids, while tuf3 encodes a protein of 391 amino acids.

Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus.

We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-1 gene encodes a protein with 71% amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr.EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.

Elongation factors in protein synthesis.

Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

Isolation and sequencing of the Alcaligenes denitrificans azurin-encoding gene: comparison with the genes encoding blue copper proteins from Pseudomonas aeruginosa and Alcaligenes faecalis.

The gene (azu) encoding azurin from Alcaligenes denitrificans has been cloned and sequenced. The gene codes for a pre-protein with a 19-aa signal peptide. Comparison with the sequences coding for the blue copper proteins from Pseudomonas aeruginosa and Alcaligenes faecalis reveals the presence of ntrA and fnr boxes in front of all three genes, instead of a regular [-10, -35]-promoter. In P. aeruginosa, the azu gene is terminated by a bidirectional terminator and flanked by open reading frames on the opposite strand.

Exact localization of the familial dysbetalipoproteinemia associated HpaI restriction site in the promoter region of the APOC1 gene.

An HpaI restriction fragment length polymorphism (RFLP) in the APOE-C1-C2 gene cluster on chromosome 19 is strongly associated with familial dysbetalipoproteinemia (type III hyperlipoproteinemia). Recently we localized this polymorphic HpaI site between the APOE and APOC1 genes. In the present paper we show by molecular cloning and sequencing that the polymorphic HpaI site is located 317 bp upstream of the transcription initiation site of the APOC1 gene. Overlapping cosmid clones allowed the construction of a detailed restriction map of the gene cluster, showing the APOC2 gene to be located 15 kb downstream of the APOC1 pseudogene.

Transcriptional control of yeast ribosomal protein synthesis during carbon-source upshift.

Shifting a yeast culture from an ethanol-based medium to a glucose-based medium causes a coordinate increase of the cellular levels of ribosomal protein mRNAs by about a factor 4 within 30 min. Making use of hybrid genes encompassing different portions of the 5'-flanking region of the L25-gene, we could show that the increase in mRNAs is a transcriptional event, mediated through DNA sequences upstream of the ribosomal protein (rp) genes. Further analysis revealed that sequence elements are involved that many rp-genes have in common and that previously were identified as transcription activation sites (RPG-boxes or UASrpg). Using appropriate deletion mutants of the fusion genes we could demonstrate that a single RPG-box is sufficient for the transcriptional upshift. In addition, both copy genes encoding rp28 which differ considerably in their extent of transcriptional activity, show the upshift effect in a proportional manner. Definite proof for the role of the UASrpg in nutritional regulation was obtained by examining the effect of a synthetic RPG-box on transcription.

Analysis of upstream activation sites of yeast ribosomal protein genes.

Transcription of the gene encoding yeast ribosomal protein L25 was previously shown to be activated through tandemly arranged upstream sequence elements that most rp-genes in yeast have in common. A single copy of such a conserved element is now demonstrated to restore transcription of an inactivated heterologous gene, which confirms its role as a genuine UAS: UASrpg. Though a single box is sufficient to activate transcription, most rp-genes harbor two neighbouring elements. Northern analysis of mutants of the L25 upstream region lacking either the gene-distal (RPG1) or the gene-proximal (RPG2) box provided evidence that RPG2 is significantly more effective than RPG1 in vivo. Moreover the sum of the effects of the individual boxes as measured separately is significantly lower than their joint effect, supporting cooperative interaction between the two boxes in vivo. Making use of oligomer-insertion experiments several additional features of the UASrpg were elucidated. First of all we confirmed that the extent of transcription activation by the UASrpg depends upon the orientation of the element. Secondly we show that a certain minimal distance (greater than 100 n) between UASrpg and the transcription initiation site is required for transcription activation. Finally, internal deletion of the L25-upstream region as well as oligomer-insertion shed some light on the nucleotide requirements of the UASrpg.

Specific binding of TUF factor to upstream activation sites of yeast ribosomal protein genes.

Transcription activation of yeast ribosomal protein genes is mediated through homologous, 12-nucleotide-long and, in general, duplicated upstream promoter elements (HOMOL1 and RPG, referred to as UASrpg). As shown previously, a yeast protein factor, TUF, interacts specifically with these conserved boxes in the 5'-flanking sequences of the elongation factor genes TEF1 and TEF2 and the ribosomal protein gene RP51A. We have now extended our studies of TUF-UASrpg binding by analysing--using footprinting and gel electrophoretic retardation techniques--the genes encoding the ribosomal proteins L25, rp28 (both copy genes), S24 + L46 and S33. Most, but not all, conserved sequence elements occurring in front of these genes, turned out to represent binding sites for the same factor, TUF. The two functionally important boxes that are found in a tandem arrangement (a characteristic of many rp genes) upstream of the L25 gene are indistinguishable in their factor binding specificity. Large differences were shown to exist in the affinity of the TUF factor for the various individual boxes and in the half-life of the protein-DNA complexes. No binding cooperativity could be demonstrated on adjacent sites on L25 or RP51A promoters. Based on binding data, the UASrpg sequence ACACCCATACAT appears to be the one recognized most efficiently by the TUF factor. Previously, no conserved box was found in front of the gene encoding S33. Nevertheless, complex formation with the protein fraction used was observed in the upstream region of the S33 gene. Competition experiments disclosed the existence of an additional binding component, distinct from TUF. This component may possibly regulate a subset of genes for the translational apparatus.

Structural and putative regulatory sequences of the gene encoding ribosomal protein L25 in Candida utilis.

Using a heterologous probe containing a fragment of the L25-gene from Saccharomyces carlsbergensis we have isolated a DNA-fragment of Candida utilis carrying the gene encoding ribosomal protein L25. This gene is present in a single copy on the C. utilis genome, though as two distinguishable alleles. Both alleles have been isolated and sequenced including their flanking regions. The nucleotide sequence of the amino acid coding region of the C. utilis gene turned out to be highly homologous (83%) to the L25-gene of S. carlsbergensis. At the protein level the degree of homology is about 87%. Codon usage in both organisms appears to be somewhat different. Just like the Saccharomyces gene, the L25 gene in C. utilis appears to be split in its 5th codon, though the identity of this codon has changed. Intron as well as 5'- and 3'-flanking sequences have almost completely diverged, with some notable exceptions. Of the intervening sequences the 5'- and 3'-splice sites as well as the putative lariat branch site are conserved. In the 5'-flanking region, at a distance of about 330 n from the initiation codon, a conserved nucleotide element is present that is very similar to the upstream transcription activation site previously found in front of the ribosomal protein genes in Saccharomyces.

Conserved sequence elements upstream of the gene encoding yeast ribosomal protein L25 are involved in transcription activation.

Previous studies have revealed the occurrence of two closely linked conserved sequence elements, designated as HOMOL 1 and RPG box, in front of most yeast ribosomal protein genes examined. To investigate whether these conserved nucleotide elements play a role in the regulation of ribosomal protein gene expression, we performed deletion analysis of the DNA region upstream of the gene encoding ribosomal protein L25. To that end we constructed a hybrid gene consisting of the pertinent 5'-flanking sequence and the Escherichia coli galK marker gene. The effects on the transcription of this fusion gene of Bal31-generated deletions were measured by Northern analysis of RNA isolated from the respective transformed yeast cells. The results demonstrate that removal of one box has a detrimental effect on the level of transcription, whereas after the deletion of both boxes hardly any transcription can be observed. Subsequently we inserted synthetic oligonucleotides in the upstream region of an L25 gene from which the original boxes had been removed. Expression of the inactivated hybrid gene turned out to be restored even by insertion of one RPG element. Moreover, the RPG box functions in both orientations, though not with equal efficiency.

Correct removal by splicing of a Neurospora intron in yeast.

Processing of intron-containing nuclear messenger RNAs in yeast require an internal conserved sequence (ICS) element, UACUAAC. Similar elements (ugCUAGAC) have been identified in sequences interrupting nuclear genes of the related ascomycete Neurospora crassa. To examine the structural splicing requirements in yeast, we constructed hybrid genes containing the intron of the Neurospora histone H3 gene and cloned them into high copy number yeast vectors. Subsequently we analyzed the RNAs transcribed in yeast from the fusion genes by Northern analysis and primer extended sequencing. It turned out that the Neurospora intron, which contains the sequence element UGCUAAC, can be removed, though very inefficiently, provided that it is located near the 5'-end of the primary transcript. This proves that an A at the second position of the ICS is no absolute requirement for splicing in yeast. In addition, the results indicate that the yeast splicing machinery is intron-position dependent.

Structure and organization of two linked ribosomal protein genes in yeast.

The genes encoding yeast ribosomal proteins rp28 and S16A are linked and occur duplicated in the yeast genome. In both gene pairs the genes are approximately 600 bp apart and are both transcribed in the same direction. Both ribosomal protein genes resemble other ribosomal protein genes studied so far in many structural aspects. The genes are interrupted by an intron near the 5'-end of their coding sequence. In addition the flanking regions contain several conserved sequence elements, which may function in transcription initiation and termination. In agreement with findings concerning other cloned yeast ribosomal protein genes, upstream homology blocks occur that may be involved in coordinate control of ribosomal protein gene transcription. The complete pattern of conserved and diverged sequences between the two duplicate gene pairs is presented.

The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences.

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.