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Y Aloni

Publications and source records attributed to Y Aloni.

At least 19 recordsLinked to original sources
PubMed2000

Pseudomembranous colitis: report of a severe case with unusual clinical signs in a young nurse.

We describe the case of a young and otherwise healthy nurse who developed pseudomembranous colitis ten days after receiving oral clindamycin for dental infection. Her clinical course was particularly stormy and was characterized by severe diarrhea and vomiting, profuse ascites, pleural effusion, abdominal tenderness, peritoneal irritation, and systemic toxicity. The Clostridium difficile assay was negative on two occasions. Features compatible with pseudomembranous colitis were seen at sigmoidoscopy, and the diagnosis was confirmed by biopsies.

Adult↗
PubMed1999

[Laparoscopic treatment of small bowel obstruction caused by adhesions].

We describe our experience in 14 patients operated on for small bowel obstruction, who underwent laparoscopic adhesiolysis. In 13 (93%) the obstruction was relieved and only 1 case required conversion to open operation. Bowel activity usually resumed within 24-48 hours, and there were no remarkable intraoperative and postoperative complications. During follow-up none developed recurrent obstruction. Our experience demonstrates that laparoscopic adhesiolysis is a valid therapeutic option. Additional experience is needed to determine which types of cases are suitable for the procedure.

Female↗
PubMed1999

Laparoscopic repair of postoperation ventral hernia. Early postoperation results.

BACKGROUND: Laparoscopic repair is a new alternative approach to postoperation ventral hernia (POVH). Whether this procedure is accompanied with acceptable operation risk and recurrence rate is not yet established. METHODS: During 1996, we performed laparoscopic repair of POVH in 53 patients. Twenty-nine (55%) of these patients had a history of at least one failed hernia repair. The size of the abdominal wall defect varied from 4 x 5 cm to 15 x 20 cm (median, 13 x 9 cm). All operations were performed with the patient under general anesthesia. In all cases, the Gore-Tex(R) Dual Mesh (W. L. Gore & Associates, Flagstaff, AZ, USA) was used in sizes varying from 5 x 7 cm to 20 x 30 cm (median, 15 x 12 cm). RESULTS: No deaths occurred as a result of the operations. Intraoperative small bowel injury occurred in two patients (3.6%), which necessitated conversion to laparotomy and performance of small bowel resection in one case and simple suture in the other. Small bowel obstruction developed during the immediate postoperation period in two patients (3.6%). In one of these patients, laparoscopic lysis of adhesions had to be performed. Graft infection with subsequent graft removal occurred in one patient (1.8%), and abdominal wall hematoma developed in another patient (1.8%). Length of hospital stay varied from 2 to 8 days (median, 3.3 days). Follow-up period ranged from 10 to 22 months (median, 17 months). During this period, recurrence of hernia occurred only in one patient in which the mesh had been removed. CONCLUSIONS: Laparoscopic repair of POVH is technically feasible. According to our experience, it is the preferred method for patients who have had an earlier failed open repair and patients in whom it is the first repair. Cases with a high likelihood for small bowel injury must be recognized and converted to routine open repair.

Adult↗
PubMed1995

Transcription elongation of the murine ornithine decarboxylase (ODC) gene is regulated in vitro at two downstream elements by different attenuation mechanisms.

Ornithine decarboxylase (ODC) plays an important role in cell proliferation. Its expression is tightly regulated at the mRNA and protein levels and is found to be deregulated in various malignancies. The rapid and dramatic induction of cellular ODC mRNA upon serum addition raised the possibility that a transcriptional attenuation mechanism may be involved in the regulation of ODC gene expression. Using transcription in HeLa nuclear extract and isolated transcription complexes, we have identified two sites of transcription arrest downstream to the transcription start site: Attenuator 1 (Att.1) located at +220, near two repeats of a USF/Myc-Max binding consensus sequence and attenuator 2 (Att.2) located at +1590 near a long stretch of T-residues. The two attenuators exhibit distinct properties as revealed by elongation of briefly initiated and partially purified transcription complexes: Att.1 serves as a transient pause site while arrest at Att.2 is more prolonged. The arrest at both attenuators is modulated by the general elongation factor TFIIS. In a promoter independent transcription system, using partially purified RNA polymerase II, only Att.2 was recognized efficiently. This suggests that the recognition of Att.2 is an intrinsic property of the polymerase while Att.1 recognition has to be facilitated by an auxiliary factor/s.

Animals↗
PubMed1994

A cellular repressor regulates transcription initiation from the minute virus of mice P38 promoter.

We previously reported that the P38 promoter of minute virus of mice (MVM) is trans activated by the viral nonstructural protein, NS1, through an interaction with a downstream promoter element designated DPE. In this communication we report the identification of a distinct downstream promoter element which inhibits transcription from the P38 promoter in vitro, in the absence of the DPE. Removal of 34 bp from the region between +95 and +129 downstream from the P38 initiation start site relieved inhibition of transcription in whole-cell extract. Inhibition was also relieved by the addition, to the transcription reaction, of excess DNA fragments which span the putative inhibiting element. This indicated the involvement of a trans-acting factor, in inhibition of transcription from the P38. Gel retardation experiments demonstrated the specific binding of a cellular protein to the inhibitory element. This P38 inhibitory element shows spacing and orientation dependence as well as promoter specificity. The regulation of viral transcription by a cellular repressor may play an important role in obtaining a fine temporal order of viral gene expression during the course of infection.

Base Sequence↗
PubMed1994

Minute virus of mice infection modifies cellular transcription elongation.

Our previous observations indicated that upon infection with minute virus of mice (MVM), Ehrlich ascites cells lose a transcription elongation activity which is essential for the readthrough of the MVM attenuator. This was monitored by the ability of extracts from uninfected but not from infected cells to support readthrough of the P4 attenuator when added to partially purified transcription elongation complexes. We have investigated the nature of this change in transcription elongation following MVM infection. In this communication, we show that infection of Ehrlich ascites cells with MVM leads to a general shift in the length of nascent mRNA synthesized in isolated nuclei and separated by sucrose gradients. Furthermore, infection leads to attenuation of transcription of the cellular gene c-fos but not c-myc. We show biochemical evidence to support a model by which, following MVM infection, there is a functional reduction in the activity of a TFIIS-like general transcriptional elongation activity.

Animals↗
PubMed1993

Histone H1-mediated inhibition of transcription initiation of methylated templates in vitro.

The detailed mechanism underlying the inhibition of transcription by DNA methylation is still obscure. Chromatin structure has frequently been proposed as a role player in this mechanism. Histone H1 is a known key element in the formation and stabilization of chromatin fibers. We describe here experiments designed to examine the effect of DNA methylation on the binding of histone H1 to DNA and the consequent inhibitory effect of the bound histone H1 on in vitro transcription. The results of these experiments showed a clear preferential binding of histone H1 to methylated DNA as compared with unmethylated DNA. The in vitro transcription assay indicated that transcription of methylated templates was inhibited at a lower histone H1/DNA ratio than of unmethylated templates, and that the extent of inhibition depends on the density of methyl groups in the promoter region. This inhibition of in vitro transcription was alleviated efficiently by methylated competitor DNA, whereas, under similar conditions, almost no effect was observed with unmethylated competitor. Experiments designed to pinpoint the stage in the transcription process that was suppressed by the preferred binding of histone H1 to methylated template revealed that inhibition occurred at the initiation and not at the elongation level.

Binding Sites↗
PubMed1993

Wild-type but not mutant p53 can repress transcription initiation in vitro by interfering with the binding of basal transcription factors to the TATA motif.

It has previously been shown that excess wild type (wt) p53 can repress the transcriptional activity of a variety of promoters in intact cells. To determine whether this transcriptional repression represented a direct effect of p53, wt and mutant p53 were prepared from E. coli-produced p53 and from insect cells infected with a recombinant baculovirus. When added into an in vitro transcription system, wt p53, but not mutant p53 reduced markedly transcription from the c-myc promoter, as well as from an array of other promoters, with the exception of an MHC class I gene promoter. The presence of wt p53 seemed to affect specifically the formation of the transcription preinitiation complex because preformed initiation complexes were completely refractory to wt p53, as was also the process of transcript elongation. Wild-type but not mutant p53 interfered with the stable binding of TBP and TFIIA to the TATA motif, although both wt and mutant p53 could associate in vitro with purified TBP. We propose that upon binding to TBP, wt but not mutant p53 specifically blocks the ability of TBP to engage in interactions required for efficient transcriptional initiation. This may account, at least in part, for the ability of excess wt p53 to inhibit cell proliferation and to interfere with neoplastic processes.

Base Sequence↗
PubMed1992

Parameters affecting the elongation block by RNA polymerase II at the SV40 attenuator 1 in vitro.

We have previously suggested that folding of the SV40 attenuator RNA 1 into two hairpin elements leads to a block of transcription elongation. Using site-directed mutagenesis, we created three templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared among the templates, in cell-free extracts. In this report, we show that at the standard temperature (30 degrees C) the elongation block occurs at elevated KCl concentrations (0.5-1.2 M KCl) while at high temperature (65 degrees C), although the transcription elongation rate increases, the block to elongation occurs at lower KCl concentrations (less than 0.5 M KCl) as well. Since under the conditions tested the extent of the elongation block is directly dependent on the stability of the hairpin structure of the RNA, as assessed by a comparison of transcription of the wild-type and mutated templates, we suggest that elevated KCl concentrations and high temperatures are favored conditions for optimizing the processes whereby the hairpin structures are recognized by the polymerase as an attenuation signal. Moreover, the present experiments indicate that cellular elongation factors are not directly involved in modulating the extent of the elongation block at the SV40 attenuator 1 in vitro. The SV40 attenuator 1 is the only eukaryotic system known to date which has been shown to have structural characteristics so similar to rho-independent terminator in prokaryotes. We discuss the similarities between the mechanisms which lead to elongation blocks at these eukaryotic and prokaryotic sites.

Base Sequence↗
PubMed1992

Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein.

Fractionation of a transcription-competent HeLa cell extract on a column containing one copy of the heptamer repeat (YSPTSPS) present in the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II resulted in the loss of transcriptional activity. Fractionation of the extracts on columns containing mutations of the heptamer repeat was without effect. Such transcriptionally inactive extracts regained their ability to specifically transcribe different class II promoters upon the addition of human TFIID, recombinant yeast TATA-binding protein (TBP), or proteins bound to the column. Fractionation of RNA polymerase II on columns containing human or yeast TBP resulted in the specific retention of the nonphosphorylated form of RNA polymerase II. The phosphorylated form of the enzyme was unable to interact with TBP. The specific interaction of RNA polymerase II with TBP was mediated by the CTD of RNA polymerase II.

Amino Acid Sequence↗
PubMed1991

A 21-base pair DNA fragment directs transcription attenuation within the simian virus 40 late leader.

Transcription through the late simian virus 40 (SV40) sequences have been examined in vivo and in vitro for identifying attenuation signals involved in regulating late RNA transcription. In addition to the previously identified and characterized attenuator 1 situated 93 nucleotides downstream from the major late transcription start site, a second attenuator, attenuator 2, situated 55 nucleotides downstream from it, has been identified. Attenuated transcripts mapping to this site have been observed in vivo as well as in several different in vitro transcription systems. The signal inducing transcription attenuation has been localized to a 21-base pair DNA fragment and has been found to function independently of the promoter directing transcription or its distance from the transcription start site. Furthermore, this attenuator, unlike that preceding it, does not include a region of dyad symmetry or A+T-rich sequences. Also, similar to the adenovirus 2 attenuator, but in contrast to SV40 attenuator 1, the block to elongation at the SV40 attenuator 2 appears to be modulated by the general transcription factors. It is concluded that in SV40 there are at least two types of attenuators: one that is dependent on RNA secondary structure and a second that is sequence specific and is modulated, at least in part, by the general transcription factors.

Animals↗
PubMed1991

Elements involved in an in vitro block to transcription elongation at the end of the L1 mRNA family of adenovirus 2.

Using the 3' end of the L1 mRNA family of adenovirus 2 (Ad2) as a model system, we investigated transcription elongation following a poly(A) signal in a cell-free system. The results show that RNA polymerase II can halt transcription elongation at a T-rich stretch in the non-coding DNA strand 20 nucleotides downstream of the poly(A) signal. The block to transcription elongation is enhanced when Sarkosyl is included in the elongation reaction. Deletion studies narrowed the region which directs the elongation block at the T-rich stretch, to an upstream fragment of 53 nucleotides that is very dA-rich and also contains a functional poly(A) signal. The deletion studies and analysis by site-directed mutagenesis indicate that in the present system, RNA secondary structure, the stretch of T's and the poly(A) signal are not the dominant elements responsible for the elongation block. The block to transcription elongation at the T-rich stretch was also shown to be 5 times more effective in an uninfected extract than in an Ad2 infected extract, which is reminiscent of the in vivo situation and is consistent with the suggestion that a trans-acting factor is involved in modulating the elongation block at the T-rich stretch.

Adenoviridae↗
PubMed1991

Transcriptional elongation by purified RNA polymerase II is blocked at the trans-activation-responsive region of human immunodeficiency virus type 1 in vitro.

It has previously been shown that the human immunodeficiency virus type 1 (HIV-1) trans-activation-responsive region (TAR) is contained in a stem-loop RNA structure. Moreover, the interaction of the RNA secondary structure with Tat, the trans-activator protein, seems to play a role in activation of transcription initiation and in preventing transcription attenuation. In this work, we have studied the ability of the HIV-1 TAR stem-loop to act as a specific attenuation signal for highly purified RNA polymerase II. We developed an in vitro system using dC-tailed DNA fragments of HIV-1 to study transcriptional control in the HIV-1 LTR. We have found that transcription in this system yields an attenuator RNA whose 3' end maps to the end of the TAR stem-loop, approximately 60 to 65 nucleotides downstream of the in vivo initiation site. Furthermore, transcription attenuation occurs only under conditions which cause displacement of the nascent transcript from the template DNA strand, thus allowing the RNA to fold into secondary structure. Evidence is provided that the purified polymerase II indeed recognizes stable RNA secondary structure as an intrinsic attenuation signal. The existence of this signal in the TAR stem-loop suggests that in vivo an antiattenuation factor, probably Tat, alone or in combination with other factors, acts to relieve the elongation block at the HIV-1 attenuation site.

Base Sequence↗
PubMed1991

Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.

Adenoviruses, Human↗
PubMed1991

The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

Animals↗
PubMed1990

In vitro regulation of human hepatitis B virus core gene transcription.

In the present study we used a HeLa whole cell extract transcription system to map the transcription start sites and the minimal promoter of the hepatitis B virus core gene. Two initiation sites located at residues 1792 +/- 5 and 1817 +/- 5 were identified. The minimal upstream region essential and sufficient for transcription was defined to a 105-base pair DNA fragment. These results are identical to the in vivo mapping of the transcription start sites and the minimal core gene promoter. When in vitro transcription elongation was carried out in the presence of the anionic detergent Sarkosyl, known to enhance premature transcription termination (attenuation), two short transcripts (as well as two run-offs) were synthesized. Kinetic studies indicated that the short transcripts resulted from a block to transcription elongation and not from RNA processing. RNA mapping showed that the short attenuated transcripts indeed initiated at the two core gene initiation sites and both prematurely terminated at nucleotide 1966 +/- 5, defined as the attenuation site. This site is located in the attenuator RNA within a uridine-rich sequence preceded by a stable hairpin structure. Attenuation at the same site occurred when transcription of the core gene was directed by the Ad2 major late promoter (MLP) and when the poly(A) signal, which precedes the attenuation site, was mutated from TATAAA to TAGAAA. We suggest that the elongation block at nt 1966 +/- 5 in vivo exerts a dual function: first, it regulates the level of RNA by attenuation during the first cycle of transcription and, second, it acts as a termination site at the end of the primary RNA transcript.

Base Sequence↗