Pieter van de Putte

The regulation of transcription initiation by integration host factor

Integration host factor (IHF) of Escherichia coli is an asymmetric histone‐like protein that binds and bends the DNA at specific sequences. IHF functions as an accessory factor in a wide variety of processes including replication, site‐specific recombination and transcription. In many of these processes IHF was shown to act as an architectural element which helps the formation of nucleo‐protein complexes by bending of the DNA at specific sites. This MicroReview shows how such a structural role of IHF can influence the initiation of transcription. In addition, it summarizes the evidence indicating that IHF can stimulate transcription via a direct interaction with RNA polymerase and explores the possibility that the asymmetry of the IHF protein might reflect such an interaction

DNA inversions in phages and bacteria

In certain phages and bacteria, there is a recombination system that specifically promotes the inversion of a DNA fragment. These inversion events appear to act as genetic switches allowing the alternate expression of different sets of genes which in general code for surface proteins. The mechanism of inversion in one class of inversion systems (Gin/Hin) has been studied in detail. It involves the formation of a highly specific nucleoprotein complex in which not only the two recombination sites and the DNA invertase participate but also a recombinational enhancer to which the DNA-bending protein Fis is bound.

AP-1 and Ets Transcription Factors Regulate the Expression of the Human SPRR1A Keratinocyte Terminal Differentiation Marker

The 173-base pair proximal promoter ofSPRR1A is necessary and sufficient for regulated expression in primary keratinocytes induced to differentiate either by increasing extracellular calcium or by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Whereas calcium-induced expression depends both on an AP-1 and an Ets binding site in this region, responsiveness to TPA resides mainly (but not exclusively) on the Ets element, indicating that Ets factors are important targets for protein kinase C signaling during keratinocyte terminal differentiation. This conclusion is further substantiated by the finding that expression of ESE-1, an Ets transcription factor involved in SPRR regulation, is also induced by TPA, with kinetics similar to SPRR1A. The strict AP-1 requirement inSPRR1A for calcium-induced differentiation is not found forSPRR2A, despite the presence of an identical AP-1 consensus binding site in this gene. Binding site swapping indicates that both the nucleotides flanking the TGAGTCA core sequence and the global promoter context are essential in determining the contribution of AP-1 factors in gene expression during keratinocyte terminal differentiation. In the distal SPRR1A promoter region, a complex arrangement of positive and negative regulatory elements, which are only conditionally needed for promoter activity, are likely involved in gene-specific fine-tuning of the expression of this member of the SPRR gene family.

On the control of transcription of bacteriophage Mu.

SummaryThe transcription pattern of bacteriophage Mu has been studied with the use of Mu-1 cts62, a thermo-inducible derivative of wild-type Mu. The rate of transcription at various times after induction was measured by pulse-labeling the RNA during synthesis and determining the fraction of Mu-specific RNA by hybridization with the separated strands of Mu-DNA. Transcription was found to take place predominantly from the heavy strand of Mu-DNA, as was found previously by Bade (1972). A study of the kinetics of this process revealed four phases. Initially after the induction the rate of transcription increases and reaches a maximum after four minutes. In the second phase during five minutes the rate falls down. During the third phase, up to 25 minutes after induction, the rate of transcription rises slowly, followed by a very rapid increase in the final phase, at the end of the lytic cycle. Phage Mu can be integrated in the host chromosome in two opposite orientations. The strand specificity, rate and time-course of transcription appeared not to be influenced by the orientation. The presence of chloramphenicol during the induction of the phage does not have an effect on the initial phase of transcription, but it prevents the decrease in the second phase. This suggests that in the early phase a Mu-specific protein is synthesized which acts as a negative regulator of trancription. In non-permissive strains, lysogenic for a phage with an amber mutation in gene A or B, the transcription during the first and the second phase is the same as with wild-type phage; in the third phase, however, there is much less transcription than with wild type phage, whereas in the final phase the increase of the transcription rate is completely absent.Control experiments showed that DNA synthesis does not take place when a non-permissive strain is infected with a phage with an amber mutation in gene A or B. Therefore we conclude that the products of the genes A and B are required, directly or indirectly, for the autonomous replication of phage DNA. Since these amber mutants are also impaired in the integration process, we conclude that the genes A and B code for regulator proteins with a crucial role in the development of bacteriophage Mu.

Cloning, characterization and DNA sequencing of the gene encoding the Mr50000 quinoprotein glucose dehydrogenase fromAcinetobacter calcoaceticus

SummaryRecently we described the cloning of the gene coding for a Mr 87000 glucose dehydrogenase (GDH-A) fromAcinetobacter calcoaceticus. In this report we describe the cloning of a gene coding for a second GDH (GDH-B) with a Mr of 50000 from the same organism. This gene was isolated using a 20-mer synthetic oligonucleotide, derived from the N-terminal amino acid sequence of purified GDH-B as a probe to screen a genomic bank. From the DNA sequence of thegdhB gene, a protein can be derived of Mr 52772 with a 24 amino acid signal peptide which is removed, resulting in the mature protein with a Mr 50231. In vitro transcription-translation of thegdhB clone shows the mature and the precursor protien. The derived amino acid sequence has no obvious homology with GDH-A ofA. calcoaceticus. We show that disaccharides are specific GDH-B substrates and that 2-deoxyglucose is specific for GDH-A.

Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.

Transcription of bacteriophage Mu

SummaryIt has previously been shown that the transcription of Mu is asymmetric and takes place on the heavy DNA strand (Bade, 1972; Wijffelman et al., 1974). The direction of transcription of Mu has now been determined by RNA-DNA hybridizations between purified Mu-RNA and the separated strands of λ-Mu hybrid phages. The direction of transcription is from the c-gene (immunity gene) end of the heavy strand to the β-end (immunity distal end) (Fig. 1). Thermo-inducible, defective Mu lysogens, in which the prophage is deleted from the β-end, have a normal early transcription pattern, but the increase of RNA at later times is absent. A defective lysogen, which contains only the immunity gene c and the genes A and B, still has an early transcription pattern similar to that of the wild-type. Therefore, we conclude that the early RNA is transcribed from that region of the Mu genome.The early Mu-RNA synthesis is negatively regulated with a minimum rate of transcription at 9 minutes after induction. Before the onset of the late RNA synthesis, at about 22 minutes there is a rather long period in which the rate of Mu-RNA synthesis slowly increases. Using DNA strands of λ-Mu hybrids which contain only that part of the Mu-DNA on which the early RNA synthesis takes place, we have determined that during the first half in the intermediate phase only early genes are transcribed.The amount of Mu-RNA synthesized by a Mu prophage carrying the X-mutation, which influences the excision of Mu, is greatly reduced. Negative regulation of early transcription occurs normally in this mutant.

Integration host factor alleviates the H-NS-mediated repression of the early promoter of bacteriophage Mu.

Integration host factor (IHF), which is a histone‐like protein, has been shown to positively regulate transcription in two different ways. It can either help the formation of a complex between a transcription factor and RNA polymerase or it can itself activate RNA polymerase without the involvement of other transcription factors. In this study, we present a third mechanism for IHF‐stimulated gene expression, by counteracting the repression by another histone‐like protein, H‐NS. The early (Pe) promoter of bacteriophage Mu is specifically inhibited by H‐NS, both in vivo and in vitro. For this inhibition, H‐NS binds to a large DNA region overlapping the Pe promoter. Binding of IHF to a binding site just upstream of Pe alleviates the H‐NS‐mediated repression of transcription. This same ihf site is also involved in the direct activation of Pe by IHF. In contrast to the direct activation by IHF, however, the alleviating effect of IHF appears not to be dependent on the relevant position of the ihf site on the DNA helix, and it also does not require the presence of the C‐terminal domain of the alpha subunit of RNA polymerase. Footprint analysis shows that binding of IHF to the ihf site destabilizes the interaction of H‐NS with the DNA, not only in the IHF‐binding region but also in the DNA regions flanking the ihf site. These results suggest that IHF disrupts a higher‐order nucleoprotein complex that is formed by H‐NS and the DNA.

Gin-mediated recombination of catenated and knotted DNA substrates: Implications for the mechanism of interaction between cis-acting sites

The Gin DNA-inversion system of bacteriophage Mu normally requires a substrate containing two inverted recombination sites (gix) and an enhancer sequence on the same supercoiled DNA molecule. The reaction mechanism was investigated by separating these sites on catenated rings. Catenanes with the gix sites on one circle and the enhancer on the other recombined efficiently. Thus, the enhancer was fully functional even though it was located in trans to the gix sites. Multiple links between the rings are required for recombination. Multiply linked catenanes with gix sites on separate circles, one of which contained the enhancer, were also efficient substrates. Knotted constructs carrying directly repeated gix sites were recombined. Catenated and knotted substrates must also be supercoiled. These experiments eliminate simple tracking or looping models as explanations for why the enhancer and gix sites must be in cis with standard substrates. Rather, the Gin synaptic complex requires the three sites to be mutually intertwined in a right-handed fashion with a unique polarity of the gix sites. This geometry is achieved by branching of the DNA substrate and requires the energy and structure of supercoiling, catenation, or knotting.

A single amino acid substitution changes the substrate specificity of quinoprotein glucose dehydrogenase in Gluconobacter oxydans

SummaryGluconobacter oxydans contains pyrroloquinoline quinone-dependent glucose dehydrogenase (GDH). Two isogenic G. oxydans strains, P1 and P2, which differ in their substrate specificity with respect to oxidation of sugars have been analysed. P1 can oxidize only d-glucose, whereas P2 is also capable of the oxidation of the disaccharide maltose. To investigate the nature of this maltose-oxidizing property we cloned the gene encoding GDH from P2. Expression of P2 gdh in P1 enables the latter strain to oxidize maltose, indicating that a mutation in the P2 gdh gene is responsible for the change in substrate specificity. This mutation could be ascribed to a 1 by substitution resulting in the replacement of His 787 by Asn.