PubMed HealthSearch

Biomedical subjects

G T Marczynski

Publications and source records attributed to G T Marczynski.

13 recordsLinked to original sources

Cell cycle control by an essential bacterial two-component signal transduction protein.

Dividing cells must coordinate cell cycle events to ensure genetic stability. Here we identify an essential two-component signal transduction protein that controls multiple events in the Caulobacter cell cycle, including cell division, stalk synthesis, and cell cycle-specific transcription. This protein, CtrA, is homologous to response regulator transcription factors and controls transcription from a group of cell cycle-regulated promoters critical for DNA replication, DNA methylation, and flagellar biogenesis. CtrA activity in the cell cycle is controlled both transcriptionally and by phosphorylation. As purified CtrA binds an essential DNA sequence motif found within its target promoters, we propose that CtrA acts in a phosphorelay signal transduction system to control bacterial cell cycle events directly at the transcriptional level.

Amino Acid Sequence

A developmentally regulated chromosomal origin of replication uses essential transcription elements.

Only one of the two chromosomes in the asymmetric Caulobacter predivisional cell initiates replication in the progeny cells. Transcription from a strong promoter within the origin occurs uniquely from the replication-competent chromosome at the stalked pole of the predivisional cell. This regulated promoter has an unusual sequence organization, and transcription from this promoter is essential for regulated (cell type-specific) replication. Our analysis defines a new class of bacterial origins and suggests a coupling between transcription and replication that is consistent with the phylogenetic relationship of Caulobacter to the ancestral mitochondrion.

Base Sequence

The control of asymmetric gene expression during Caulobacter cell differentiation.

The dimorphic bacterium Caulobacter crescentus provides a simple model for cellular differentiation. Each cell division produces two distinct cell types: a swarmer cell and a stalked cell. These cells possess distinct functional morphologies and differential programs of transcription and DNA replication. The synthesis of a single polar flagellum is restricted to the swarmer pole of the predivisional cell by a genetic hierarchy comprising at least 50 genes whose transcription is regulated by novel and ubiquitous promoters, cognate sigma factors, and auxiliary transcriptional regulators. Chromosome replication is restricted to the stalked cell by a unique chromosome origin of replication that may be regulated by a novel cell-specific transcriptional control system. Phosphorylation signals, DNA methylation, differential chromosome structures, protein targeting, and selective protein degradation are also involved in establishing and maintaining cellular asymmetry. The molecular details of these universal cellular processes in C. crescentus will provide paradigms applicable to many general aspects of cellular differentiation.

Bacterial Proteins

Gal4 protein binding is required but not sufficient for derepression and induction of GAL2 expression.

The Saccharomyces cerevisiae GAL2 gene upstream activator sequence (UAS) region was examined for protein bound in vivo by chromatin footprinting at high resolution. Gal4 transcriptional activator protein binds to the two consensus UAS sites whether GAL2 expression is induced, uninduced, or repressed by growth with different carbon sources. Although wild type strains show loss of the Gal4 protein-specific footprint in repressing media containing glucose, constitutive high level expression of Gal4 protein restores the GAL2 UAS footprints without fully derepressing GAL2 transcription. Thus binding of the Gal4 activator to target sites in the DNA is required but not sufficient for GAL2 derepression and induction. Gal4-independent protein-DNA complexes were also detected in the region, including one over the previously noted centromere-binding protein (CP1) site upstream of the Gal4 complexes.

Base Sequence

Bacterial chromosome origins of replication.

Bacteria regulate chromosomal replication from one specific origin. We compare the regulatory requirements, DNA structures, and biochemical properties of the prototypic Escherichia coli origin with those of evolutionarily distant Bacillus subtilis and Caulobacter crescentus origins. The ubiquitous DnaA protein is a major regulator of all three bacterial origins. Unique features of these origins, however, may reflect specific regulatory requirements placed on them.

Bacteria

Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus.

Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.

Bacterial Proteins

Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle.

Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.

Cell Cycle

Regulatory interactions between phospholipid synthesis and DNA replication in Caulobacter crescentus.

Several Caulobacter crescentus mutants with lesions in phospholipid biosynthesis have DNA replication phenotypes. A C. crescentus mutant deficient in glycerol 3-phosphate dehydrogenase activity (gpsA) blocks phospholipid synthesis, ceases DNA replication, and loses viability in the absence of a glycerol phosphate supplement. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. The first effect of withholding supplement was the cessation of synthesis of phosphatidylglycerol, a major component of the C. crescentus membrane. In the absence of glycerol 3-phosphate, DNA replication was initiated in the stalked cell at the correct time in the cell cycle and at the correct site on the chromosome. However, after replication proceeded bidirectionally for a short time, DNA synthesis dropped to a low level. The cell cycle blocked at a distinct middivision stalked cell, and this was followed by cell death. The "glycerol-less" death of the gpsA mutant could be prevented if the cells were treated with novobiocin to prevent the initiation of DNA replication. Our observations suggest that the processivity of C. crescentus replication requires concomitant phospholipid synthesis and that cell death results from incomplete replication of the chromosome.

Bacteria

Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested.

Base Sequence

A transcription map of a yeast centromere plasmid: unexpected transcripts and altered gene expression.

YCp19 is a yeast centromere plasmid capable of autonomous replication in both yeast and E. coli (J. Mol. Biol., 158: 157-179, 1982). It is stably maintained as a single copy in the yeast cell and is therefore a model yeast "minichromosome" and cloning vector. We have located the positions and measured the abundance of the in vivo yeast transcripts from YCp19. Transcripts from the selectable marker genes TRP1 and URA3 were present at increased levels relative to chromosomal copies of the genes. Unanticipated transcripts from the yeast CEN4 and E. coli pBR322 sequences were also found. Although much of the plasmid vector is actively transcribed in vivo, the regions around the most useful cloning sites (BamHI, EcoRI, SalI) are free of transcripts. We have analyzed transcription of BamHI inserts containing promoter variants of the HIS3 gene and determined that although initiation events are accurate, plasmid context may alter levels of gene expression.

Centromere

Algorithm for calculating theoretical probabilities of patterns generated by sequential inequality testing.

Temporal patterns of extracellularly monitored single neuronal impulses or 'spike' trains can be viewed as stochastic point processes that carry information from one neuron to another. There are indications that the dependencies among sequential spike intervals, if treated as sequential inequality patterns, encompass much more than 7 spike intervals. Hence, to fill the gap between the available knowledge and the experimental need, a limited stochastic model of inequality patterns was reviewed and its inherent symmetries were explored. The symmetric attributes of the model, based on three through seven spike intervals, led to an algorithm which allows one to readily compute the theoretical distribution of inequality patterns of considerable complexity and length suitable for studying neuronal responses and other phenomena.

Action Potentials

Visual attention and neuronal firing patterns in the feline pulvinar nucleus of thalamus.

In behaving cats, temporal patterns of neuronal firing were studied during slow wave sleep (SWS), motionless quiet wakefulness (QW) coupled with specific direction of the animal's attention, and during bar pressing performance (BP) for milk reward. The analysis was based on relative relations between sequential spike intervals. The strength of the method is based on the fact that the probabilities of occurrence of patterns are determined by the history of a spike train. During SWS, the neuronal firing modes closely followed the theoretical model of independent distribution of intervals, whereas during QW and BP specific for each neuron departures from the model, i.e., patterning was observed. Most importantly, in seven chronically studied neurons idiosyncratic patterns were related to direction of the animal's attention, and, very likely, to the visual forms the animals gazed at, because the patterns disappeared in the dark and during SWS without major changes in the mean firing rate. The replications of patterns upon recurrence of a particular direction of attention was proven statistically. The constancy and idiosyncrasy of these patterns were apparent even though the comparable episodes occurred several hours apart, and the animals slept and/or ate in between, and the distance, i.e., the retinal size of visual forms varied from one episode to another. On the basis of correlative evidence, it was argued that, compared to more abstract modes of information processing, the identification and quantification of patterns based on relative relations between intervals require the least amount of storage of intermediate results. Hence, these patterns are likely to represent a simple and phylogenetically old principle of communication between neurons. It was postulated that the idiosyncrasy and invariance of patterns may play a role in constancy of feature extraction and Gestalt perception.

Animals

Neuronal firing patterns in the feline hippocampus during sleep and wakefulness.

The study addressed the problem of information transmission in mammalian brain as reflected in the emergence or disappearance of temporal patterns in extracellularly monitored single action potentials from the dorsal hippocampus of unrestrained cats during slow wave sleep (SWS), rapid eye movement sleep (REM), and motionless quiet wakefulness (QW). The spike trains were analyzed with a nonparametric technique. Chi-square statistics were used to measure deviation of firing patterns from the theoretical model which is based on the assumption that the intervals are random and/or independent from each other. The plots of the chi-square values for a given set of patterns represented the neuronal 'signatures' characteristic of a behavioral state. During SWS most neurons followed the theoretical model, i.e. their 'signatures' were flat and statistically non-significant. However, during REM sleep and QW their firing modes showed specific deviations from the theoretical model: some patterns occurred more often while others less often than expected, thus generating large and statistically significant 'signatures'. During REM sleep some neurons shared similar tendencies in their departures from the theoretical model. However, during QW the same neurons developed their individual 'signatures' which were significantly different from each other. Hence, the QW episodes were characterized by a greater differentiation of neuronal firing patterns. The mean firing rate and the shape of the time interval histogram were not necessarily correlated with the emergence of specific temporal patterns in spike trains. The results suggest that information transmission from one neuron to another depends on the emergence of repetitive and specific temporal patterns. The strong tendency of most neurons to lapse during SWS into a firing mode that closely follows the theoretical model constitutes the basis for a working hypothesis which states that the essence of SWS recovery in cognitive systems is the disappearance of temporal patterns, and that the 'noisy' interactions between neurons plays an important role in the recuperative processes.

Animals