The Bacillus subtilis SftA (YtpS) and SpoIIIE DNA translocases play distinct roles in growing cells to ensure faithful chromosome partitioning

In several bacterial species, the faithful completion of chromosome partitioning is known to be promoted by a conserved family of DNA translocases that includes Escherichia coli FtsK and Bacillus subtilis SpoIIIE. FtsK localizes at nascent division sites during every cell cycle and stimulates chromosome decatenation and the resolution of chromosome dimers formed by recA -dependent homologous recombination. In contrast, SpoIIIE localizes at sites where cells have divided and trapped chromosomal DNA in the membrane, which happens during spore development and under some conditions when DNA replication is perturbed. SpoIIIE completes chromosome segregation post-septationally by translocating trapped DNA across the membrane. Unlike E. coli , B. subtilis contains a second uncharacterized FtsK/SpoIIIE-like protein, SftA (formerly YtpS). We report that SftA plays a role similar to FtsK during each cell cycle but cannot substitute for SpoIIIE in rescuing trapped chromosomes. SftA colocalizes with FtsZ at nascent division sites but not with SpoIIIE at sites of chromosome trapping. SftA mutants divide over unsegregated chromosomes more frequently than wild-type unless recA is inactivated, suggesting that SftA, like FtsK, stimulates chromosome dimer resolution. Having two FtsK/SpoIIIE paralogues is not conserved among endospore-forming bacteria, but is highly conserved within several groups of soil- and plant-associated bacteria.     

Density Transfer Studies of DNA Isolated from BACILLUS SUBTILIS after Exposure to Phenethyl Alcohol

The aim of this study was to determine whether there are specific weak points in the Bacillus subtilis chromosome and if so whether the replication point is the site of breakage. To answer these questions, B. subtilis chromosomes were partially labeled with 5-bromodeoxyuridine (5-BUdR). Sheared or unsheared preparations of partially labeled chromosomes which may or may not contain replication forks were analyzed for the distribution of genetic markers in a CsCl density gradient. Two sets of experiments based upon the density transfer experiments of Yoshikawa and Sueoka (1963) were performed: (1) experiments in which the origin of the chromosome was labeled and (2) experiments in which the terminus of the chromosome was labeled. In the first experiment, strain 23 (thy(-), his(-)) spores were germinated in the presence of 5-BUdR for various lengths of time and then transferred to fresh medium containing phenethyl alcohol (PEA) and thymidine (TdR). The DNA was isolated before and after transfer to PEA and TdR. In the second experiment strain 23 (thy(-), his(-)) spores were germinated in the presence of TdR and then PEA was added. After various lengths of time transfer was made to fresh medium containing PEA and 5-BUdR. The DNA was extracted by an extremely gentle technique to avoid breakage and centrifuged in a CsCl density gradient. PEA was added to the germinated spores to prevent dichotomous replication, but PEA did not prevent dichotomous replication in any of these experiments. This contradicts the conclusion of others that PEA prevents the chromosome from entering a new round of replication, but allows the chromosome to complete the round of replication already begun. The following observations offer support for the hypothesis that the replication point is a weak point in the chromosome: (1) when conditions were created to obtain partially labeled chromosomes with replication points: (a) labeled markers appeared at the hybrid density, (b) unlabeled markers appeared at the light density, (c) shearing of the DNA had little effect on the CsCl density gradient, except on a small proportion of labeled markers which had not appeared at the hybrid density prior to shearing; (2) when conditions were created to obtain partially labeled chromosomes with no replication points: (a) the majority of DNA molecules appeared at an intermediate density between the hybrid and the light densities, (b) the labeled and unlabeled markers appeared in the intermediate peak with approximately the same ratio as in the DNA preparations, (c) the labeled markers were found in the intermediate peak except where dichotomous replication had occurred, (d) after shearing, the labeled markers appeared at the hybrid density and the unlabeled markers appeared at the light density. Thus it is concluded that the replication point is a weak point in the B. subtilis chromosome where breakage easily occurs.     

Nonrandom Sister Chromatid Segregation and Nuclear Migration in Hyphae of Aspergillus nidulans

Radioactive conidiospores of Aspergillus nidulans were prepared by growing a purine-requiring mutant with tritiated adenine. When these spores germinated in a nonradioactive medium, the dispersion of the original chromosome set could be followed by treating the hyphae with ribonuclease and preparing radioautograms. Germinating spores with four or eight nuclei contained two highly labeled nuclei and two or six nuclei with much less or no radioactivity. Successive mitotic divisions thus distributed the deoxyribonucleic acid (DNA) of the eight spore chromosomes among only two of the progeny nuclei. The two nuclei containing the original chromosome set were not dispersed at random along the linear hypha but were usually located near the growing tip. These results are compatible with the view that chromatids containing DNA strands of identical age segregate as a unit during mitosis. They further indicate that the mechanism which disperses newly formed nuclei in the growing hypha can distinguish between nuclei containing DNA strands of different ages.     

Chromosome strand segregation during sporulation in Bacillus subtilis

After the initiation of spore formation in Bacillus subtilis, the products of the final round of DNA replication segregate into two cells, i.e. the prespore and the mother cell. The prespore, which is known to contain a single completed chromosome, develops into a mature endospore which can be readily separated from mother cells and non-sporulating cells on the basis of its resistance properties. We have used a procedure originally developed to label the terminus region of the B. subtilis chromosome to specifically label the newly synthesized strands of DNA during the final round of DNA replication before sporulation. We have purified prespore DNA and used strand-specific probes to measure the radioactivity incorporated. The results show that the sister chromosomes segregate at random into the prespore. This result has implications for the segregation of chromosomes during vegetative growth and for the generation of cellular asymmetry during sporulation.     

Chromosome segregation in an asymmetrically dividing bacterium,Caulobacter crescentus

The mode of chromosome segregation in an asymmetrically dividing bacterium, Caulobacter crescentus, was studied by examining the fate of labeled DNA strands. Swarmer cells (one type of Caulobacter daughter cell), in which single strands of DNA had been labeled with [³H]thymidine during the previous round of chromosome replication, were grown synchronously in a non-radioactive medium for two generations. The distribution of radioactivity among the cells was visualized by autoradiography under a phase-contrast microscope. The labeled DNA strands in each cell were found to consist of two conserved units. From this, we propose a model in which the swarmer cell has two identical chromosomes, which are segregated into the progeny swarmer cell and the progeny stalked cell after chromosome replication.     

Physical mapping of the CC-chemokine gene cluster on the human 17q11. 2 region.

Chemokines are a family of small secreted proteins that are involved in the trafficking of leukocytes by acting on G-protein-coupled receptors. Specific chemokines are also implicated in the regulation of angiogenesis and mobilization of hematopoietic cell precursors. Chemokines are subdivided into four groups on the basis of the relative positions of their conserved cysteines. For the CC-chemokine group, in which the first two (of four) conserved cysteines are adjacent, 22 members have been described so far. In this work, we have analyzed the genomic organization of these genes. We first assigned the genes encoding CC-chemokines to chromosomal regions and organized their relative positioning by using two radiation hybrid panels. Fifteen CC-chemokine genes were shown to be clustered within the 17q11.2 region of the human genome. These genes appeared to be segregated into two subclusters separated by about 2. 25 Mb (9 cR). Contigs of bacterial artificial chromosomes (BAC) covering these two subclusters were subsequently isolated and the localizations of the CC-chemokine genes within these contigs determined. The relative positioning of the BAC clones was determined with the help of fluorescence hybridization on combed genomic DNA. The cluster organization of the various CC-chemokine genes in the genome was found to be grossly consistent with their structural similarities. This map of the CC-chemokine gene cluster should facilitate the determination of the full sequence of the chromosomal region.     

Nonselective Incorporation into Sporangium of Either “Older” or “Younger” Chromosome of the Vegetative Cell During Sporulation in Bacillus cereus

Employing Bacillus cereus strain 2, we examined the fate of two chromosomes contained in vegetative cells in the course of sporulation. Cytological observations and quantitative estimation of deoxyribonucleic acid (DNA) confirmed the earlier observations that, during the course of sporulation, one of two chromosomes of the vegetative cell was incorporated into the sporangium and the other disappeared into the medium as the result of cell lysis. Log-phase cells, labeled completely with thymine-2-(14)C in the presence of deoxyadenosine, were cultured in the "cold" glucose-glutamate-glycine-salts medium, and culture samples, taken at intervals at successive generations, were subjected to sporulation in glutamate-salts medium. The percentage of radioactivity in the spores separated from each culture remained almost unchanged at nearly 50% and was independent of the number of generations of the preceding culture in the "cold" medium. This suggests that the selective incorporation into the sporangium of either the "older" or "younger" chromosome of a vegetative cell does not occur in the course of spore formation. Some examples of the selective and nonselective behavior of DNA molecules in cellular events in microorganisms are cited.     

Segregation of Bacillus subtilis chromosomes radioactively labeled during the first round of replication after germination of spores.

Spores of Bacillus subtilis W23 thy his were allowed to incorporate [3H]thymine for short periods of time either continuously from, or soon after, the start of the first round of replication after germination. They were then transferred to nonradioactive medium to allow growth into microcolonies (up to 12 cells), which were autoradiographed. The relative numbers of various types (major versus minor) of grain clusters associated with individual microcolonies throughout the populations were scored. Analysis of the results showed clearly that, in the majority of spores at least, only one chromosome was undergoing replication soon after the start of deoxyribonucleic acid synthesis. Furthermore, under the conditions used, no evidence for initiation of replication of a second chromosome within 25 min after the first could be obtained. Accepting that B. subtilis spores are essentially homogenous in deoxyribonucleic acid content, the results support the conclusion that the spore contains only one copy of the chromosome, not two.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan<Emphasis Type="Italic"> Hydra</Emphasis>

To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8–10 m) containing about 3×10⁹ bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5–10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.Abbreviations CT Chromosome territory - BrdU Bromodeoxyuridine - IdU Iododeoxyuridine - CldU Chlorodeoxyuridine Communicated by E.A. Nigg     

Identifying conserved gene clusters in the presence of homology families.

The study of conserved gene clusters is important for understanding the forces behind genome organization and evolution, as well as the function of individual genes or gene groups. In this paper, we present a new model and algorithm for identifying conserved gene clusters from pairwise genome comparison. This generalizes a recent model called "gene teams." A gene team is a set of genes that appear homologously in two or more species, possibly in a different order yet with the distance of adjacent genes in the team for each chromosome always no more than a certain threshold. We remove the constraint in the original model that each gene must have a unique occurrence in each chromosome and thus allow the analysis on complex prokaryotic or eukaryotic genomes with extensive paralogs. Our algorithm analyzes a pair of chromosomes in O(mn) time and uses O(m+n) space, where m and n are the number of genes in the respective chromosomes. We demonstrate the utility of our methods by studying two bacterial genomes, E. coli K-12 and B. subtilis. Many of the teams identified by our algorithm correlate with documented E. coli operons, while several others match predicted operons, previously suggested by computational techniques. Our implementation and data are publicly available at euler.slu.edu/ approximately goldwasser/homologyteams/.     

Highly specific labeling of the Bacillus subtilis chromosome terminus

By making use of the sporulation process, the terminus region of the Bacillus subtilis chromosome has been labeled with [3H]thymine in a highly specific manner. The result achieved supports the view that B. subtilis spores contain only completed chromosomes.     

RING-POSITION AND FREQUENCY OF CHIASMA FAILURE IN RHOEO SPATHACEA

The mean chiasma number per cell was 11.44 ± 0.04 in 489 cells. Due to chiasma failures, the ring-of-twelve chromosomes may be broken into two or three chains. Cells with four or more chains were not observed. All six possible two-chain situations and eight different threechain situations were found. All possible lengths of chains from one to all twelve chromosomes were found, with “chain”-of-one inordinately frequent. The overall mean number of chromosomes in 273 chains in 188 cells is 8.26 ± 0.31 and 5.38 ± 0.31 among 154 chains in the 69 cells that had two or more chains. The mean number of chains per cell among these 188 was 1.45 ± 0.13. In 73 cells, 113 chiasma failures were found to be distributed at random among the twelve chromosome arm positions. The absence of association either between length of arm or between presence-absence of secondary constriction and frequency of chiasma failure support the generally accepted theory that, in Rhoeo, synapsis and crossing over are restricted to small terminal segments on all chromosomes.     

Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during spore germination.

During germination of spores of Bacillus species the degradation of the spore's pool of small, acid-soluble proteins (SASP) is initiated by a protease termed GPR, the product of the gpr gene. Bacillus megaterium and B. subtilis mutants with an inactivated gpr gene grew, sporulated, and triggered spore germination as did gpr+ strains. However, SASP degradation was very slow during germination of gpr mutant spores, and in rich media the time taken for spores to return to vegetative growth (defined as outgrowth) was much longer in gpr than in gpr+ spores. Not surprisingly, gpr spores had much lower rates of RNA and protein synthesis during outgrowth than did gpr+ spores, although both types of spores had similar levels of ATP. The rapid decrease in the number of negative supertwists in plasmid DNA seen during germination of gpr+ spores was also much slower in gpr spores. Additionally, UV irradiation of gpr B. subtilis spores early in germination generated significant amounts of spore photoproduct and only small amounts of thymine dimers (TT); in contrast UV irradiation of germinated gpr+ spores generated almost no spore photoproduct and three to four times more TT. Consequently, germinated gpr spores were more UV resistant than germinated gpr+ spores. Strikingly, the slow outgrowth phenotype of B. subtilis gpr spores was suppressed by the absence of major alpha/beta-type SASP. These data suggest that (i) alpha/beta-type SASP remain bound to much, although not all, of the chromosome in germinated gpr spores; (ii) the alpha/beta-type SASP bound to the chromosome in gpr spores alter this DNA's topology and UV photochemistry; and (iii) the presence of alpha/beta-type SASP on the chromosome is detrimental to normal spore outgrowth.     

Genetic Mapping in Saccharomyces IV. Mapping of Temperature-Sensitive Genes and Use of Disomic Strains in Localizing Genes

Through use of tetrad, random spore, trisomic, and mitotic analysis procedures a large number of genes, including 48 new genetic markers, were studied for their locations on the genetic maps of the yeast Saccharomyces cerevisiae. Eighteen new centromere linked genes were discovered and all but one was located on various ones of the 16 previously-established chromosomes. Five fragments of linked genes were also assigned to chromosomes; four were located on known chromosomes while the fifth determined one arm of a new chromosome. The experiments indicate that seventeen is likely to be the haploid chromosome number in this yeast. Most chromosomes have been established by genetic means to be metacentric and their genetic lengths vary from 5 cM to approximately 400 cM. Functionally-related sets of genes generally were found to be dispersed over the genome.     

Conservation of relative chromosome positioning in normal and cancer cells

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM(-/-) mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.     

Genome-wide characterization of tetrahymena thermophila chromosome breakage sites. I. Cloning and identification of functional sites

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.     

The ordered arrangement of chromosomes in the Chinese hamster spermatocyte nucleus

Summary The question of chromosome distribution in the mammalian nucleus is addressed, and data are provided in support of the ordered arrangement of chromosomes in the Chinese hamster spermatocyte. Testicular cells were dispersed and air-dried without prior fixation, then stained and karyotyped. The position of chromosome telomeres in 217 pachytene spermatocytes was determined in relation to four concentric rings which equally divided the nuclear area. The distribution of telomeres showed a progressive decline from the central to the peripheral rings. This was particularly pronounced for chromosomes 1–7, but was reversed for the XY chromosomes. The distribution of the total as well as of the individual chromosomes was significantly different from that expected on the basis of random distribution. The only exceptions to this were chromosomes 8–10, which exhibited random distribution. Thus, while chromosomes 1–7 had a central position, the XY pair had a peripheral localization. The mean ring position appeared to be related to chromosome length, except for the XY chromosomes, suggesting that chromosome length may determine chromosome position.