Griet Van Houwe

Copurification of E. coli RNAase E and PNPase: Evidence for a specific association between two enzymes important in RNA processing and degradation

Ribonuclease E (RNAase E) was isolated in a complex that also contained polynucleotide phosphorylase (PNPase). Besides copurification, evidence for an association of these enzymes comes from sedimentation and immunoprecipitation experiments. Highly purified RNAase E correctly processed E. coli 5S ribosomal RNA, bacteriophage T4 gene 32 mRNA and E. coli ompA mRNA at sites known to depend on the rne gene for cleavage in vivo. The difference between previous smaller estimates of the size of RNAase E and that reported here apparently is due to the sensitivity of the enzyme to proteolysis during purification. The discovery of a specific association between RNAase E and PNPase raises the intriguing possibility that these enzymes act cooperatively in the processing and degradation of RNA.

Chromatin boundaries in budding yeast: the nuclear pore connection.

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

Nuclear pore association confers optimal expression levels for an inducible yeast gene.

The organization of the nucleus into subcompartments creates microenvironments that are thought to facilitate distinct nuclear functions. In budding yeast, regions of silent chromatin, such as those at telomeres and mating-type loci, cluster at the nuclear envelope creating zones that favour gene repression. Other reports indicate that gene transcription occurs at the nuclear periphery, apparently owing to association of the gene with nuclear pore complexes. Here we report that transcriptional activation of a subtelomeric gene, HXK1 (hexokinase isoenzyme 1), by growth on a non-glucose carbon source led to its relocalization to nuclear pores. This relocation required the 3′ untranslated region (UTR), which is essential for efficient messenger RNA processing and export, consistent with an accompanying report. However, activation of HXK1 by an alternative pathway based on the transactivator VP16 moved the locus away from the nuclear periphery and abrogated the normal induction of HXK1 by galactose. Notably, when we interfered with HXK1 localization by either antagonizing or promoting association with the pore, transcript levels were reduced or enhanced, respectively. From this we conclude that nuclear position has an active role in determining optimal gene expression levels.

Live Imaging of Telomeres: yKu and Sir Proteins Define Redundant Telomere-Anchoring Pathways in Yeast

BACKGROUND The positioning of chromosomal domains within interphase nuclei is thought to facilitate transcriptional repression in yeast. Although this is particularly well characterized for telomeres, the molecular basis of their specific subnuclear organization is poorly understood. The use of live fluorescence imaging overcomes limitations of in situ staining on fixed cells and permits the analysis of chromatin dynamics in relation to stages of the cell cycle. RESULTS We have characterized the dynamics of yeast telomeres and their associated domains of silent chromatin by using rapid time-lapse microscopy. In interphase, native telomeres are highly dynamic but remain within a restricted volume adjacent to the nuclear envelope. This constraint is lost during mitosis. A quantitative analysis of selected mutants shows that the yKu complex is necessary for anchoring some telomeres at the nuclear envelope (NE), whereas the myosin-like proteins Mlp1 and Mlp2 are not. We are able to correlate increased telomeric repression with increased anchoring and show that silent chromatin is tethered to the NE in a Sir-dependent manner in the absence of the yKu complex. Sir-mediated anchoring is S phase specific, while the yKu-mediated pathway functions throughout interphase. Subtelomeric elements of yeast telomere structure influence the relative importance of the yKu- and Sir-dependent mechanisms. CONCLUSIONS Interphase positioning of telomeres can be achieved through two partially redundant mechanisms. One requires the heterodimeric yKu complex, but not Mlp1 and Mlp2. The second requires Silent information regulators, correlates with transcriptional repression, and is specific to S phase.

Chromosome looping in yeast: telomere pairing and coordinated movement reflect anchoring efficiency and territorial organization

Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes.

Genetic identification of cloned fragments of bacteriophage T4 DNA and complementation by some clones containing early T4 genes

SummaryBacteriophage T4 DNA containing cytosine has been obtained from cells infected with phage mutant in genes 42, 56,denA anddenB. This DNA can be cut by a number of restriction endonucleases. Fragments obtained by digestion of this DNA withEcoRI have been cloned using the vector plasmid pCR1.Clones containing T4 DNA were identified by hybridization with radioactive early and late T4 RNA. A simple marker rescue technique is described for the genetic identification of the cloned T4 fragments. Some of the T4-hybrid plasmids which contain entire T4 genes can complement temperature sensitive and amber mutants of T4.

Bacteriophage T4 gene transcription studied by hybridization to cloned restriction fragments.

Cloned restriction fragments of bacteriophage T4 DNA (Mattson et al. , 1977; Selzer et al. , 1978) have been used as hybridization probes to study T4 transcription during infection of Escherichia coli . Eleven early genes and about 35 late genes have been studied. The time at which the genes are active, and their rate of transcription, have been measured by quantitative hybridization to excess DNA immobilized on nitrocellulose filters. The early genes studied include representatives of the three pre-replicative classes previously defined: immediate early (IE), delayed early (DE), and quasi-late (QL). Transcription of genes known or inferred to be in the IE class (genes 30, 39, 52, 40 and/or 41 and 42 ) are transcribed at the highest relative rate during the first four minutes of infection, and are transcribed in the presence of chloramphenicol. Transcription of DE genes rIIA, rIIB and 43 is highest four to eight minutes after infection and does not occur in the presence of chloramphenicol. Transcription of rIIB can be detected prior to transcription of rIIA , confirming previous data (Schmidt et al. , 1970) on rII transcription. The only genes studied which exhibit quasi-late behavior are the transfer RNA genes. They also have IE characteristics. Both l - and r -strand transcripts have been detected from the late region of the chromosome. Some of the l -strand (early) transcription detected from the late region may be anti-late RNA, but it is also possible that there are early genes in one of these regions (between genes 24 and 25 ). Differences in the kinetics of late gene transcription have been observed. A comparison of the earliest times at which late gene transcriptions can be detected shows that initiation of late gene transcription is not entirely synchronous. For example, transcription of genes 50 to 6 is detected by six minutes but transcription of genes 12 to 16 is not detected until 10 minutes. For all of the late genes studied, transcription, once begun, continues until at least 24 minutes. However, some genes ( 50 to 6 ) are transcribed at their maximal relative rate by 12 minutes whereas the relative rate of transcription of other genes ( 12 to 16, 23 ) continues to increase until at least 20 minutes. The relative rates of synthesis of late gene transcripts vary much less than the relative rates of synthesis of late proteins, suggesting that differential synthetic rates of late proteins are not controlled predominantly by messenger RNA synthetic rates.

Subtelomeric factors antagonize telomere anchoring and Tel1-independent telomere length regulation

Yeast telomeres are anchored at the nuclear envelope (NE) through redundant pathways that require the telomere‐binding factors yKu and Sir4. Significant variation is observed in the efficiency with which different telomeres are anchored, however, suggesting that other forces influence this interaction. Here, we show that subtelomeric elements and the insulator factors that bind them antagonize the association of telomeres with the NE. This is detectable when the redundancy in anchoring pathways is compromised. Remarkably, these same conditions lead to a reduction in steady‐state telomere length in the absence of the ATM‐kinase homologue Tel1. Both the delocalization of telomeres and reduction in telomere length can be induced by targeting of Tbf1 or Reb1, or the viral transactivator VP16, to a site 23 kb away from the TG repeat. This correlation suggests that telomere anchoring and a Tel1‐independent pathway of telomere length regulation are linked, lending a functional significance to the association of yeast telomeres with the NE.

Control of synthesis of glucosyl transferase and lysozyme messengers after T4 infection

Abstract Messenger RNA from T4-infected cells is able to direct the cell-free synthesis of the early phage enzyme, α-glucosyl transferase (Young, 1970) and the late enzyme, T4 lysozyme (Salser, Gesteland & Bolle, 1967). We have used in vitro protein synthesis programmed by RNA from T4-infected cells as an assay to study the in vivo synthesis of the messengers for these enzymes. α-Glucosyl transferase messenger RNA which is functional in vitro can first be detected in vivo at about four minutes after infection at 30 °C. The amount of messenger increases rapidly until about ten minutes after infection and then decays with a half-life of about three minutes. This latter observation suggests that transferase messenger RNA synthesis is repressed at ten minutes. During infection of the non-permissive host with a DNA negative amber mutant, amN122 , transferase messenger RNA synthesis is prolonged several minutes so that more messenger accumulates than during infection of the permissive host or during an infection with wild type T4. The increased messenger level can account for the hyper-production of T4 early enzymes observed after infection with DNA-negative mutants. During infection with a mutant defective in gene 55 (necessary for maturation) both the synthesis and the degradation of the transferase messenger proceed normally, indicating that neither the gene 55 product nor any of the late T4 proteins that are absent in gene 55-infected cells are necessary for the repression of transcription of the transferase cistron. Neither mutant makes a detectable amount of functional lysozyme messenger RNA in the non-permissive host.

In vivo expression of the rII region of bacteriophage T4 present in chimeric plasmids

SummaryThe expression of the T4 rII genes in uninfected cells has been examined by use of recombinant plasmids. Hybridization analysis of pulse-labelled RNA prepared from cells carrying pTB101, a plasmid that contains the end of T4 gene 60 and the beginning of gene rIIA, shows that about 0.7% of the labelled RNA is rII specific. By contrast, only 0.02% of pulse-labelled RNA prepared from cells carrying plasmid pTB301, which probably contains the middle-mode rIIB promoter, may be rII specific. When separated strands of T4 DNA were used for hybridization, we found that the pTB101 transcripts have a strand specificity identical to that of the rIIA transcripts made during phage infection. The same strand specificity was observed irrespective of the orientation of the inserted DNA in the vector. This result argues that the transcripts initiate within the inserted DNA rather than somewhere else on the plasmid. We also found that essentially none of the pulse-labelled pTB101 RNA would hybridize to the DNA of a T4 deletion mutant that lacks the rIIA gene. This suggests that little of the gene 60 DNA of the plasmid is being transcribed. In addition to the rII transcript, a new protein of 56,000 Daltons molecular weight is found in cells carrying pTB101. Fingerprint analysis of the protein shows that it is specified by the rIIA gene of the plasmid. Taken together, these results indicate that transcription of the plasmid rIIA gene initiates at or near the beginning of the gene.