Biomedical subjects

D B Krimer

Publications and source records attributed to D B Krimer.

At least 19 recordsLinked to original sources

PubMed2003

Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells.

Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24-48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.

Acetamides↗

PubMed2002

DNA knotting caused by head-on collision of transcription and replication.

Collision of transcription and replication is uncommon, but the reason for nature to avoid this type of collision is still poorly understood. In Escherichia coli pBR322 is unstable and rapidly lost without selective pressure. Stability can be rescued if transcription of the tetracycline-resistance gene (Tet(R)), progressing against replication, is avoided. We investigated the topological consequences of the collision of transcription and replication in pBR322-derived plasmids where head-on collision between the replication fork and the RNA polymerase transcribing the Tet(R) gene was allowed or avoided. The results obtained indicate that this type of collision triggers knotting of the daughter duplexes behind the fork. We propose this deleterious topological consequence could explain the instability of pBR322 and could be also one of the reasons for nature to avoid head-on collision of transcription and replication.

Autoradiography↗

PubMed2002

Supercoiling, knotting and replication fork reversal in partially replicated plasmids.

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

Blotting, Southern↗

PubMed2002

Knotting dynamics during DNA replication.

The topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two-dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.

DNA Replication↗

PubMed2000

Premature termination of DNA replication in plasmids carrying two inversely oriented ColE1 origins.

In Escherichia coli plasmids carrying two inversely oriented ColE1 origins, DNA replication initiates at only one of the two potential origins. The other silent origin acts as a replication fork barrier. Whether this barrier is permanent or simply a pausing site remains unknown. Here, we used a repeated primer extension assay to map in vivo, at the nucleotide level, the 5' end of the nascent strand where initiation and blockage of replication forks occurs. Initiation occurred primarily at the previously defined origin, however, an alternative initiation site was detected 17 bp upstream. At the barrier, the lagging strand also terminated at the main initiation site. Therefore, the 5' end of the nascent strand at the barrier was identical to that generated during initiation. This observation strongly suggests that blockage of the replication fork at the silent origin is not just a pausing site but permanent, and leads to a premature termination event.

Bacteriocin Plasmids↗

PubMed2000

Bi-directional replication and random termination.

Two-dimensional (2D) agarose gel electrophoresis was used to study termination of DNA replication in a shuttle vector, YRp7', when it replicated in Escherichia coli, Saccharomyces cerevisiae and Xenopus egg extracts. In E. coli, the 2D gel patterns obtained were consistent with uni-directional replication initiated at a specific site, the ColE1 origin. In consequence, termination also occurred precisely at the ColE1 origin. In Xenopus egg extracts, the particular shape of the bubble arc as well as the triangular smear detected to the left of the simple-Y pattern indicated random initiation and termination. In S.cerevisiae, initiation occurred at the ARS1 origin and replication proceeded in a bi-directional manner. However, termination did not always occur at a specific site 180 degrees across from the origin, but almost all along the south hemisphere of the plasmid. Inversion, deletion or replacement of DNA sequences located throughout this hemisphere did not eliminate random termination. Analysis of the replication intermediates of another yeast plasmid bearing a different origin, ARS305, also exhibited random termination. We propose that the random termination events observed in S.cerevisiae could be due to an asynchronous departure of both forks from the bi-directional origin in addition to differences in the rate of fork progression. These observations could be extended to all bi-directional origins.

Animals↗

PubMed2000

Visualisation of plasmid replication intermediates containing reversed forks.

Blockage of replication forks can have deleterious consequences for the cell as it may prompt premature termination of DNA replication. Moreover, the blocked replication intermediate (RI) could be particularly sensitive to recombination processes. We analysed the different populations of RIs generated in vivo in the bacterial plasmid pPI21 after pausing of replication forks at the inversely oriented ColE1 origin. To achieve this goal, a new method was developed based on two-dimensional agarose gel electrophoresis. This method allows the isolation of specific RIs, even when they were rather scarce, from the total DNA. Here we describe the occurrence of RI restriction fragments containing reversed forks. These Holliday-like structures have been postulated but never observed before.

DNA↗

PubMed1999

Formation of knots in partially replicated DNA molecules.

Bacterial plasmids with two origins of replication in convergent orientation are frequently knotted in vivo. The knots formed are localised within the newly replicated DNA regions. Here, we analyse DNA knots tied within replication bubbles of such plasmids, and observe that the knots formed show predominantly positive signs of crossings. We propose that helical winding of replication bubbles in vivo leads to topoisomerase-mediated formation of knots on partially replicated DNA molecules.

DNA Replication↗

PubMed1999

Characterization of the pea rDNA replication fork barrier: putative cis-acting and trans-acting factors.

It was previously shown that in pea (Pisum sativum), rDNA repeats contain a polar replication fork barrier that blocks progression of the replication machinery moving in the direction opposite to transcription. This barrier maps in the untranscribed spacer close to the 3' end of the 25S gene. Very similar barriers are also found in the rDNA of yeast, Xenopus and mammalian cultured cells. This high conservation indicates that the rDNA barrier plays a relevant biological role. Progression of replication forks through the DNA sequence where the barrier maps in pea was investigated in plasmids replicating in Escherichia coli and Saccharomyces cerevisiae. No barrier was detected in these heterologous systems, indicating that the DNA sequence by itself was insufficient to block the replication machinery. Therefore, trans-acting factors were likely to be required. Taking advantage of the natural sequence heterogeneity in pea rDNA, we obtained evidence that a 27 bp imperfect tandem repeat is involved in the arrest of replication. Moreover, nuclear protein(s) specifically bound to this repeat suggesting that this DNA/protein complex is responsible for the polar arrest of replication forks.

Base Sequence↗

PubMed1998

DnaB helicase is unable to dissociate RNA-DNA hybrids. Its implication in the polar pausing of replication forks at ColE1 origins.

A series of plasmids were constructed containing two unidirectional ColE1 replication origins in either the same or opposite orientations and their replication mode was investigated using two-dimensional agarose gel electrophoresis. The results obtained showed that, in these plasmids, initiation of DNA replication occurred at only one of the two potential origins per replication round regardless of origins orientation. In those plasmids with inversely oriented origins, the silent origin act as a polar pausing site for the replication fork initiated at the other origin. The distance between origins (up to 5.8 kilobase pairs) affected neither the interference between them to initiate replication nor the pausing function of the silent origin. A deletion analysis indicated that the presence of a transcription promoter upstream of the origin was the only essential requirement for it to initiate replication as well as to account for its polar pausing function. Finally, in vitro helicase assays showed that Escherichia coli DnaB is able to melt DNA-DNA homoduplexes but is very inefficient to unwind RNA-DNA hybrids. Altogether, these observations strongly suggest that replication forks pause at silent ColE1 origins due to the inability of DnaB helicase, which leads the replication fork in vivo, to unwind RNA-DNA hybrids.

Autoradiography↗

PubMed1998

A computer model for the analysis of DNA replication intermediates by two-dimensional agarose gel electrophoresis.

We present a computer model to predict the patterns expected for the replication intermediates (RIs) of DNA fragments analyzed by neutral/neutral two-dimensional (2D) agarose gel electrophoresis. The model relies on the mode of replication (uni- or bi-directional), the electrophoretic mobility of linear DNA fragments and the retardation caused by the three-dimensional shape of non-linear molecules. The utility of this model is demonstrated with two examples: replication analysis of the plasmids pBR322 and pHH5.8 in Escherichia coli after digestions with EcoRI and HindIII, respectively.

Computer Simulation↗

PubMed1998

Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis.

Neutral/neutral two-dimensional (2D) agarose gelelectrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide to account for any possible changes in the migration behavior of DNA molecules caused by intercalation of this planar agent. Finally, DNA samples were isolated from a recA-strain of Escherichia coli , as well as after direct labeling of the replication intermediates in extracts of Xenopus laevis eggs. Altogether, the results obtained demonstrated that 2D gels can be readily used to identify most of the complex topological populations that circular molecules can adopt in vivo in both bacteria and eukaryotic cells.

Animals↗

PubMed1998

Co-localization of polar replication fork barriers and rRNA transcription terminators in mouse rDNA.

We investigated the replication of the region where transcription terminates in mouse rDNA. It contains a replication fork barrier (RFB) that behaves in a polar manner, arresting only replication forks moving in the direction opposite to transcription. This RFB consists of several closely spaced fork arrest sites that co-localize with the transcription terminator elements, known as Sal boxes. Sal boxes are the target for mTTF-I (murine transcription termination factor I). These results suggest that both termination of rRNA transcription and replication fork arrest may share cis-acting as well as trans-acting factors.

Animals↗

PubMed1997

Cloning, sequencing and expression in MEL cells of a cDNA encoding the mouse ribosomal protein S5.

We describe the isolation and characterization of a cDNA encoding the mouse S5 ribosomal protein. It was isolated from a MEL (murine erythroleukemia) cell cDNA library by differential hybridization as a down regulated sequence during HMBA-induced differentiation. Northern series analysis showed that S5 mRNA expression is reduced 5-fold throughout the differentiation process. The mouse S5 mRNA is 760 bp long and encodes for a 204 amino acid protein with 94% homology with the human and rat S5.

Amino Acid Sequence↗

PubMed1997

H3.3A variant histone mRNA containing an alpha-globin insertion: modulated expression during mouse gametogenesis correlates with meiotic onset.

Replacement-variant H3.3 histones have been isolated and sequenced in different eukaryotes, but no functional H3.3A gene has been characterized in the mouse so far. We have cloned an H3.3A cDNA from a mouse fetal ovary library, differentially screened with testis versus somatic cDNA probes. We showed this gene contains a region homologous to the reverse and complementary alpha-globin gene. We believe such a structure could have been generated by retroposition during the evolution of both globin and histone gene families. The sequence coding for H3.3A is 76.6% homologous to the mouse H3.3B gene at the nucleotide level and differs in only one amino acid at the protein level. The high degree of homology between these genes and the H3.3 variant histones from other eukaryotes reveals the conservation of these replication-independent class of histones throughout evolution. Analysis of gene expression reveals a developmental regulation concurrent with meiotic progression, with the highest level of transcript detection coincident with meiotic onset during both oogenesis and spermatogenesis.

Amino Acid Sequence↗

PubMed1997

Developmental expression of H3.3A variant histone mRNA in mouse.

Cloning and characterization of H3.3A variant histone expression has recently been reported to be associated with meiotic development in mouse testis and ovary. Using Northern analysis and in situ hybridization, the pattern of H3.3A expression was studied during the development of different tissues. In addition to the differential expression detected in male and female meiosis, H3.3A was found to be highly expressed in preantral follicles of adult ovaries and in the basal regions of seminiferous epithelium corresponding to spermatogonia. Different patterns of expression were observed in somatic tissues, which also differed with respect to the developmental stage of the tissue. The lowest expression was detected in adult skeletal muscle. High expressions were found in foetal liver and spinal cord. These different expressions might reflect a possible function of H3.3A in cell differentiation as detected in MEL cells.

Animals↗

PubMed1996

The ColE1 unidirectional origin acts as a polar replication fork pausing site.

Co-orientation of replication origins is the most common organization found in nature for multimeric plasmids. Streptococcus pyogenes broad-host-range plasmid pSM19035 and Escherichia coli pPI21 are among the exceptions. pPI21, which is a derivative of pSM19035 and pBR322, has two long inverted repeats, each one containing a potentially active ColE1 unidirectional origin. Analysis of pPI21 replication intermediates (RIs) by two-dimensional agarose gel electrophoresis and electron microscopy revealed the accumulation of a specific RI containing a single internal bubble. The data obtained demonstrated that initiation of DNA replication occurred at a single origin in pPI21. Progression of the replicating fork initiated at either of the two potential origins was transiently stalled at the other inversely oriented silent ColE1 origin of the plasmid. The accumulated RIs, containing an internal bubble, occurred as a series of stereoisomers with different numbers of knots in their replicated portion. These observations provide one of the first functional explanations for the disadvantage of head-to-head plasmid multimers with respect to head-to-tail ones.

Bacteriocin Plasmids↗

PubMed1993

Induction of H3.3 replacement histone mRNAs during the precommitment period of murine erythroleukemia cell differentiation.

Differential hybridization to a cDNA library made from the mRNA of differentiating mouse erythroleukemia (MEL) cells has been used to identify sequences that are induced during the early stages of MEL cell differentiation. One of the differentially expressed genes identified encodes the H3.3 histone subtype. We show here that the three polyadenylated mRNAs produced from the H3.3B gene, as well as the single mRNA produced from the related H3.3A gene, are coordinately induced during the first few hours of MEL cell differentiation and subsequently down regulated as cells undergo terminal differentiation. Nuclear run-on transcription experiments indicate that the accumulation and decay of these mRNAs are controlled at the post-transcriptional level. Unlike the polyadenylated mRNAs of two H1 histone genes that exhibit similar kinetics of induction and decay controlled by c-myc, induction of the H3.3 mRNAs is unaffected by deregulated expression of c-myc.

Animals↗