This study investigates what happens to proteins associated with DNA during chromosome translocation in Bacillus subtilis sporulation. Using fluorescent protein fusions and a mutant that forms two forespores, the authors show that RNA polymerase, chromosome remodeling proteins, and transcription factors are stripped off the chromosome as it is translocated into the forespores. Specifically, they demonstrate that a TetR-GFP fusion bound to an operator array is efficiently removed from the translocating DNA. Additionally, in vitro experiments indicate that the ATPase domain of SpoIIIE can displace RNA polymerase from DNA. These results suggest that SpoIIIE translocates naked DNA and strips associated proteins during chromosome transport, which may play a role in reprogramming gene
Wiki glossary epigenetic control of gene expressionBárbara Pérez
This document is a glossary defining terms related to epigenetics and gene expression. It defines over 80 key terms concisely, including definitions for adenosine triphosphate, alleles, autosomes, Barr body, bisulfite sequencing, blastocyst, boundary element, central dogma, chromatin, chromosome, cytosine methylation, DNA methylation, epigenetic reprogramming, epigenetics, embryonic stem cells, euchromatin, gene, genomic imprinting, histones, homologous chromosomes, imprinted genes, meiosis, mitosis, mRNA, nucleosome, placenta, and pluripotent cells. The glossary provides succinct yet informative definitions for fundamental concepts in molecular biology
This document summarizes recent evidence that the arrangement of chromosomes, gene loci, and nuclear bodies within the cell nucleus is not random but rather exhibits spatial organization that influences gene expression and nuclear processes. Techniques such as fluorescence in situ hybridization and chromosome conformation capture have provided insights into the positioning of chromosomes and genes relative to nuclear landmarks. Chromosomes occupy distinct territories within the nucleus and preferentially localize near the nuclear interior or periphery depending on their gene content. Association with nuclear subcompartments such as the nuclear lamina, nuclear pores, nucleoli, and polycomb bodies can impact the transcriptional state of genes and chromatin domains. Advances in genome-wide and time-lapse imaging approaches are helping to further characterize nuclear organization
The document traces the evolution of the concept of the gene from its earliest conceptions in the early 20th century to more modern understandings. It notes that early thinkers like Mendel, Garrod, and Morgan viewed the gene as the basic unit of heredity controlling phenotypic traits. Later work established that genes encode enzymes and polypeptides, with hypotheses like the one gene-one enzyme hypothesis. Still more recent work determined that genes are sequences of DNA that code for polypeptide chains. The document also discusses different types of genes and models of gene action like the operon model.
This document summarizes maternal inheritance of chloroplast DNA. It discusses how chloroplasts are inherited in the variegated four o' clock plant Mirabilis jalapa. Flowers on green branches produce only green offspring, while flowers on white branches produce only white offspring, demonstrating maternal inheritance of chloroplasts. Flowers on variegated branches produce offspring with mixed phenotypes, due to segregation of chloroplast types in the egg cell cytoplasm. This cytoplasmic inheritance demonstrates that in some plants, chloroplast DNA is inherited maternally rather than paternally.
This document discusses gene function and the relationship between genes and proteins. It begins by explaining the one-gene-one-enzyme hypothesis proposed by Beadle and Tatum, which demonstrated that mutations in specific genes resulted in defects in single enzymes. The document then discusses protein structure and how amino acid sequences encode higher-level structures. It explores how gene mutations can result in single amino acid changes in proteins. Finally, it discusses how genes control cellular metabolism and provides examples of genetic diseases caused by enzyme deficiencies.
This presentation elaborates regarding introduction to genetics, chromosomes, DNA, RNA, Genetics of developmental disorders of teeth, Genetics of craniofacial disorders and syndromes, genetics of cleft lip and palate, malocclusion and dental caries
This document provides an overview of genetics and heredity. It begins with an introduction to genetics and heredity. It then discusses Mendel's experiments with pea plants which formed the basis of genetics and led to his laws of inheritance. The document describes cell structure and the cell cycle, including mitosis and meiosis. It explains DNA, chromosomes, genes, transcription and translation. The human genome project is also mentioned. Overall, the document covers the key concepts and history of genetics from its early discoveries to modern understanding of inheritance and DNA.
Wiki glossary epigenetic control of gene expressionBárbara Pérez
This document is a glossary defining terms related to epigenetics and gene expression. It defines over 80 key terms concisely, including definitions for adenosine triphosphate, alleles, autosomes, Barr body, bisulfite sequencing, blastocyst, boundary element, central dogma, chromatin, chromosome, cytosine methylation, DNA methylation, epigenetic reprogramming, epigenetics, embryonic stem cells, euchromatin, gene, genomic imprinting, histones, homologous chromosomes, imprinted genes, meiosis, mitosis, mRNA, nucleosome, placenta, and pluripotent cells. The glossary provides succinct yet informative definitions for fundamental concepts in molecular biology
This document summarizes recent evidence that the arrangement of chromosomes, gene loci, and nuclear bodies within the cell nucleus is not random but rather exhibits spatial organization that influences gene expression and nuclear processes. Techniques such as fluorescence in situ hybridization and chromosome conformation capture have provided insights into the positioning of chromosomes and genes relative to nuclear landmarks. Chromosomes occupy distinct territories within the nucleus and preferentially localize near the nuclear interior or periphery depending on their gene content. Association with nuclear subcompartments such as the nuclear lamina, nuclear pores, nucleoli, and polycomb bodies can impact the transcriptional state of genes and chromatin domains. Advances in genome-wide and time-lapse imaging approaches are helping to further characterize nuclear organization
The document traces the evolution of the concept of the gene from its earliest conceptions in the early 20th century to more modern understandings. It notes that early thinkers like Mendel, Garrod, and Morgan viewed the gene as the basic unit of heredity controlling phenotypic traits. Later work established that genes encode enzymes and polypeptides, with hypotheses like the one gene-one enzyme hypothesis. Still more recent work determined that genes are sequences of DNA that code for polypeptide chains. The document also discusses different types of genes and models of gene action like the operon model.
This document summarizes maternal inheritance of chloroplast DNA. It discusses how chloroplasts are inherited in the variegated four o' clock plant Mirabilis jalapa. Flowers on green branches produce only green offspring, while flowers on white branches produce only white offspring, demonstrating maternal inheritance of chloroplasts. Flowers on variegated branches produce offspring with mixed phenotypes, due to segregation of chloroplast types in the egg cell cytoplasm. This cytoplasmic inheritance demonstrates that in some plants, chloroplast DNA is inherited maternally rather than paternally.
This document discusses gene function and the relationship between genes and proteins. It begins by explaining the one-gene-one-enzyme hypothesis proposed by Beadle and Tatum, which demonstrated that mutations in specific genes resulted in defects in single enzymes. The document then discusses protein structure and how amino acid sequences encode higher-level structures. It explores how gene mutations can result in single amino acid changes in proteins. Finally, it discusses how genes control cellular metabolism and provides examples of genetic diseases caused by enzyme deficiencies.
This presentation elaborates regarding introduction to genetics, chromosomes, DNA, RNA, Genetics of developmental disorders of teeth, Genetics of craniofacial disorders and syndromes, genetics of cleft lip and palate, malocclusion and dental caries
This document provides an overview of genetics and heredity. It begins with an introduction to genetics and heredity. It then discusses Mendel's experiments with pea plants which formed the basis of genetics and led to his laws of inheritance. The document describes cell structure and the cell cycle, including mitosis and meiosis. It explains DNA, chromosomes, genes, transcription and translation. The human genome project is also mentioned. Overall, the document covers the key concepts and history of genetics from its early discoveries to modern understanding of inheritance and DNA.
1. The document summarizes key events and discoveries in genetics, including Mendel's work with pea plants in the 1860s, the identification of DNA as the genetic material in the 1940s, and the development of techniques like recombinant DNA and PCR in the 1970s-1980s.
2. It describes the central dogma of molecular biology whereby DNA is transcribed into RNA which is then translated into protein. The processes of transcription and translation are explained in detail.
3. The three main principles of Mendelian inheritance are summarized: dominance, segregation, and independent assortment. The document also provides examples to illustrate these principles.
This document discusses different concepts of genes including classical concepts like alleles and modern concepts like jumping genes, overlapping genes, split genes, nested genes, and fusion genes. It then focuses on jumping genes or transposons, providing details on their discovery by Barbara McClintock in maize in the 1940s. Transposons are able to move from one location in the chromosome to another. The document discusses types of transposons like IS-elements, P-elements, and retrotransposons. It also discusses the significance of transposons and examples of transposon systems in maize like the Ac-Ds system.
The document discusses various concepts related to genetics and genes. It defines key terms like DNA, mRNA, gene, coding region, exons, introns, regulatory sequences, alleles, loci, genotype, phenotype, traits, karyotype and more. It explains genes are segments of DNA that encode instructions to make proteins, while regulatory sequences control gene expression. mRNA carries gene copies from DNA in the nucleus to cellular machinery for protein production.
CYTOPLASMIC INHERITANCE WITH REFERENCETO MITOCHONDRIAL INHERITANCE IN YEASTBishnuPatra1
This document summarizes a presentation on cytoplasmic inheritance with reference to mitochondrial inheritance in yeast. It discusses how cytoplasmic inheritance transmits genes outside the nucleus, usually from the female parent. Mitochondria contain their own circular DNA called mt-DNA. Mitochondrial inheritance refers to traits encoded in the mitochondrial genome being inherited, generally from the female parent. The document uses petite mutations in yeast mitochondria as an example, describing three types of petite mutants: 1) Segregational petite mutants created by nuclear mutations that segregate mendelianly, 2) Neutral petite mutants whose offspring are wild type when crossed with wild type yeast, and 3) Suppressive petite mutants that dominate and suppress wild type mitochondrial function in offspring
Genomic imprinting refers to genes whose expression depends on whether they are inherited maternally or paternally. Imprinted genes are regulated by epigenetic mechanisms like DNA methylation and histone modifications. Disruption of imprinting can cause diseases in both humans and animals. In humans, imprinting disorders include Prader-Willi and Angelman syndromes, which result from deletions on chromosome 15 and can be paternal or maternal in origin. In animals, disruption of imprinting can cause conditions like large offspring syndrome.
This document discusses the structure of genes in prokaryotes and eukaryotes. It defines a gene as a sequence of DNA that codes for a specific protein. Prokaryotic genes are continuous with three regions - a promoter, coding sequence, and terminator. Eukaryotic genes can be complex with introns and exons, promoters, and regulatory elements. Key differences are that prokaryotic genes lack introns while eukaryotic genes undergo splicing to remove introns. The document also outlines several characteristics of genes like their transmission from parents to offspring and ability to mutate.
1) Beadle and Tatum's experiment using bread mold Neurospora showed that each gene encodes the structure of one polypeptide, supporting the "one gene, one enzyme" hypothesis.
2) Meselson and Stahl's experiment using radioisotope labeling of DNA demonstrated that DNA replication occurs via the semi-conservative model, where each new DNA molecule contains one original and one newly synthesized strand.
This document provides information on organelle DNA, specifically mitochondrial DNA (mtDNA). It discusses the structure and function of mitochondria and cells. It describes how mtDNA is thought to originate from engulfed bacteria in early eukaryotic cells based on the endosymbiotic theory. MtDNA is maternally inherited, exists in high copy number, and lacks recombination. It has a higher mutation rate than nuclear DNA. MtDNA mutations can cause mitochondrial diseases if a tissue-specific threshold of mutations is reached.
Plasmids are small, circular DNA molecules that are self-replicating and carried by bacteria. They range in size from 2-100kb and can contain genes for antibiotic resistance. Bacterial genomes exist as a single circular chromosome that is highly condensed and packaged. Viruses have RNA or DNA genomes that are either single or double-stranded. Their genomes must be able to be recognized and expressed by their host cell. Mitochondria and chloroplasts originated from endosymbiotic bacteria and contain their own genomes that are maternally inherited and range in size and structure between species. Plant mitochondrial DNA can be much larger than animals.
The document discusses pedigree and genes, including the chance that three children will be heterozygous if their parents are heterozygous for a gene. It then provides information on pedigrees, dominant and recessive traits, and how traits are passed down based on whether alleles are dominant or recessive. The document also discusses chromosomes, including their structure, number and size in different species. It summarizes the early discoveries around chromosomes and genes, including linkage of genes to chromosomes through experiments in fruit flies. Overall, the document provides a high-level overview of heredity through pedigrees and genes and their relationship to chromosome structure.
Molecular study of Bacteria in relation to heredity and variationNilakshiKakati1
The document discusses heredity and variation in bacteria through molecular studies. It covers various genetic elements in bacteria like the chromosome, plasmids, bacteriophages, and transposable elements. It also describes different types of mutations that cause genetic variation, and mechanisms of gene transfer between bacteria like transformation, transduction, and conjugation. Fluctuation tests demonstrated that variation in bacteria is due to pre-existing mutations rather than environmental changes.
1. Fred Griffith discovered that a substance present in the virulent S strain of Streptococcus pneumoniae could permanently transform the nonlethal R strain into the deadly S strain.
2. Avery, MacLeod, and McCarty identified this "transforming principle" as DNA, providing the first evidence that DNA serves as the genetic material.
3. Hershey and Chase used radioactively labeled proteins and DNA from T2 viruses to infect E. coli cells. They found that most of the radioactively labeled DNA entered the bacterial cells while most of the labeled proteins remained outside, demonstrating that the genetic material of viruses is DNA rather than protein.
Cytoplasmic or non-nuclear inheritance involves the transmission of genes located outside the nucleus in organelles like chloroplasts and mitochondria. There are several mechanisms of cytoplasmic inheritance in seed plants and gymnosperms. In angiosperms, mechanisms include exclusion or degradation of organelles in the generative or sperm cells. Studies using techniques like DAPI staining and Southern blotting validated that cytoplasmic DNA is lost during pollen maturation in maternally inherited plants. Cytoplasmic inheritance is significant for traits like cytoplasmic male sterility and generation of novel varieties by organelle-specific mutagens.
This document discusses several genetics concepts and experiments including: coiling of shells in snails and their dextral and sinistral forms, the kappa particle found in Paramecium studied by T.M. Sonneborn in 1938, leaf variegation patterns in Mirabilis jalapa plants, and types of male sterility including genetic male sterility.
This document provides an overview of genetics and evolution through genetics concepts. It discusses key concepts like natural selection, genes, chromosomes, DNA, RNA, mutations, and how traits are passed down from one generation to the next through cell division processes like mitosis and meiosis. It also summarizes DNA structure and function, how DNA is replicated, and how proteins are synthesized through transcription and translation of genetic code.
FtsK is a DNA translocase in E. coli that is involved in chromosome dimer resolution and segregation. Through single-molecule experiments tracking FtsK movement on DNA substrates containing the E. coli chromosome region near the dif site, the authors identified four zones where FtsK frequently reverses direction of translocation. Analysis of these zones identified the DNA sequence motif GNGNAGGG as a candidate sequence that causes FtsK to pause and reverse direction upon encountering it from the 3' end of the G-rich strand. FtsK paused for an average of 1.0 seconds at these reversal sites.
This document summarizes research on the DNA translocase SpoIIIE in Bacillus subtilis. The key findings are:
1) SpoIIIE transports DNA directionally from the mother cell to the forespore during sporulation.
2) The g-domain of SpoIIIE is necessary for establishing directionality of DNA transport in vivo, but not for mechanical translocation in vitro.
3) SpoIIIE recognizes specific DNA sequences called SRS that are highly skewed on the B. subtilis chromosome, similar to how the related protein FtsK recognizes KOPS sequences. Interaction with SRS sequences regulates the direction of SpoIIIE-mediated DNA transport.
1. The document summarizes key events and discoveries in genetics, including Mendel's work with pea plants in the 1860s, the identification of DNA as the genetic material in the 1940s, and the development of techniques like recombinant DNA and PCR in the 1970s-1980s.
2. It describes the central dogma of molecular biology whereby DNA is transcribed into RNA which is then translated into protein. The processes of transcription and translation are explained in detail.
3. The three main principles of Mendelian inheritance are summarized: dominance, segregation, and independent assortment. The document also provides examples to illustrate these principles.
This document discusses different concepts of genes including classical concepts like alleles and modern concepts like jumping genes, overlapping genes, split genes, nested genes, and fusion genes. It then focuses on jumping genes or transposons, providing details on their discovery by Barbara McClintock in maize in the 1940s. Transposons are able to move from one location in the chromosome to another. The document discusses types of transposons like IS-elements, P-elements, and retrotransposons. It also discusses the significance of transposons and examples of transposon systems in maize like the Ac-Ds system.
The document discusses various concepts related to genetics and genes. It defines key terms like DNA, mRNA, gene, coding region, exons, introns, regulatory sequences, alleles, loci, genotype, phenotype, traits, karyotype and more. It explains genes are segments of DNA that encode instructions to make proteins, while regulatory sequences control gene expression. mRNA carries gene copies from DNA in the nucleus to cellular machinery for protein production.
CYTOPLASMIC INHERITANCE WITH REFERENCETO MITOCHONDRIAL INHERITANCE IN YEASTBishnuPatra1
This document summarizes a presentation on cytoplasmic inheritance with reference to mitochondrial inheritance in yeast. It discusses how cytoplasmic inheritance transmits genes outside the nucleus, usually from the female parent. Mitochondria contain their own circular DNA called mt-DNA. Mitochondrial inheritance refers to traits encoded in the mitochondrial genome being inherited, generally from the female parent. The document uses petite mutations in yeast mitochondria as an example, describing three types of petite mutants: 1) Segregational petite mutants created by nuclear mutations that segregate mendelianly, 2) Neutral petite mutants whose offspring are wild type when crossed with wild type yeast, and 3) Suppressive petite mutants that dominate and suppress wild type mitochondrial function in offspring
Genomic imprinting refers to genes whose expression depends on whether they are inherited maternally or paternally. Imprinted genes are regulated by epigenetic mechanisms like DNA methylation and histone modifications. Disruption of imprinting can cause diseases in both humans and animals. In humans, imprinting disorders include Prader-Willi and Angelman syndromes, which result from deletions on chromosome 15 and can be paternal or maternal in origin. In animals, disruption of imprinting can cause conditions like large offspring syndrome.
This document discusses the structure of genes in prokaryotes and eukaryotes. It defines a gene as a sequence of DNA that codes for a specific protein. Prokaryotic genes are continuous with three regions - a promoter, coding sequence, and terminator. Eukaryotic genes can be complex with introns and exons, promoters, and regulatory elements. Key differences are that prokaryotic genes lack introns while eukaryotic genes undergo splicing to remove introns. The document also outlines several characteristics of genes like their transmission from parents to offspring and ability to mutate.
1) Beadle and Tatum's experiment using bread mold Neurospora showed that each gene encodes the structure of one polypeptide, supporting the "one gene, one enzyme" hypothesis.
2) Meselson and Stahl's experiment using radioisotope labeling of DNA demonstrated that DNA replication occurs via the semi-conservative model, where each new DNA molecule contains one original and one newly synthesized strand.
This document provides information on organelle DNA, specifically mitochondrial DNA (mtDNA). It discusses the structure and function of mitochondria and cells. It describes how mtDNA is thought to originate from engulfed bacteria in early eukaryotic cells based on the endosymbiotic theory. MtDNA is maternally inherited, exists in high copy number, and lacks recombination. It has a higher mutation rate than nuclear DNA. MtDNA mutations can cause mitochondrial diseases if a tissue-specific threshold of mutations is reached.
Plasmids are small, circular DNA molecules that are self-replicating and carried by bacteria. They range in size from 2-100kb and can contain genes for antibiotic resistance. Bacterial genomes exist as a single circular chromosome that is highly condensed and packaged. Viruses have RNA or DNA genomes that are either single or double-stranded. Their genomes must be able to be recognized and expressed by their host cell. Mitochondria and chloroplasts originated from endosymbiotic bacteria and contain their own genomes that are maternally inherited and range in size and structure between species. Plant mitochondrial DNA can be much larger than animals.
The document discusses pedigree and genes, including the chance that three children will be heterozygous if their parents are heterozygous for a gene. It then provides information on pedigrees, dominant and recessive traits, and how traits are passed down based on whether alleles are dominant or recessive. The document also discusses chromosomes, including their structure, number and size in different species. It summarizes the early discoveries around chromosomes and genes, including linkage of genes to chromosomes through experiments in fruit flies. Overall, the document provides a high-level overview of heredity through pedigrees and genes and their relationship to chromosome structure.
Molecular study of Bacteria in relation to heredity and variationNilakshiKakati1
The document discusses heredity and variation in bacteria through molecular studies. It covers various genetic elements in bacteria like the chromosome, plasmids, bacteriophages, and transposable elements. It also describes different types of mutations that cause genetic variation, and mechanisms of gene transfer between bacteria like transformation, transduction, and conjugation. Fluctuation tests demonstrated that variation in bacteria is due to pre-existing mutations rather than environmental changes.
1. Fred Griffith discovered that a substance present in the virulent S strain of Streptococcus pneumoniae could permanently transform the nonlethal R strain into the deadly S strain.
2. Avery, MacLeod, and McCarty identified this "transforming principle" as DNA, providing the first evidence that DNA serves as the genetic material.
3. Hershey and Chase used radioactively labeled proteins and DNA from T2 viruses to infect E. coli cells. They found that most of the radioactively labeled DNA entered the bacterial cells while most of the labeled proteins remained outside, demonstrating that the genetic material of viruses is DNA rather than protein.
Cytoplasmic or non-nuclear inheritance involves the transmission of genes located outside the nucleus in organelles like chloroplasts and mitochondria. There are several mechanisms of cytoplasmic inheritance in seed plants and gymnosperms. In angiosperms, mechanisms include exclusion or degradation of organelles in the generative or sperm cells. Studies using techniques like DAPI staining and Southern blotting validated that cytoplasmic DNA is lost during pollen maturation in maternally inherited plants. Cytoplasmic inheritance is significant for traits like cytoplasmic male sterility and generation of novel varieties by organelle-specific mutagens.
This document discusses several genetics concepts and experiments including: coiling of shells in snails and their dextral and sinistral forms, the kappa particle found in Paramecium studied by T.M. Sonneborn in 1938, leaf variegation patterns in Mirabilis jalapa plants, and types of male sterility including genetic male sterility.
This document provides an overview of genetics and evolution through genetics concepts. It discusses key concepts like natural selection, genes, chromosomes, DNA, RNA, mutations, and how traits are passed down from one generation to the next through cell division processes like mitosis and meiosis. It also summarizes DNA structure and function, how DNA is replicated, and how proteins are synthesized through transcription and translation of genetic code.
FtsK is a DNA translocase in E. coli that is involved in chromosome dimer resolution and segregation. Through single-molecule experiments tracking FtsK movement on DNA substrates containing the E. coli chromosome region near the dif site, the authors identified four zones where FtsK frequently reverses direction of translocation. Analysis of these zones identified the DNA sequence motif GNGNAGGG as a candidate sequence that causes FtsK to pause and reverse direction upon encountering it from the 3' end of the G-rich strand. FtsK paused for an average of 1.0 seconds at these reversal sites.
This document summarizes research on the DNA translocase SpoIIIE in Bacillus subtilis. The key findings are:
1) SpoIIIE transports DNA directionally from the mother cell to the forespore during sporulation.
2) The g-domain of SpoIIIE is necessary for establishing directionality of DNA transport in vivo, but not for mechanical translocation in vitro.
3) SpoIIIE recognizes specific DNA sequences called SRS that are highly skewed on the B. subtilis chromosome, similar to how the related protein FtsK recognizes KOPS sequences. Interaction with SRS sequences regulates the direction of SpoIIIE-mediated DNA transport.
This document summarizes research identifying the sequence recognition domain of the bacterial DNA translocase FtsK. Through experiments using truncated versions of FtsK, the researchers determined that the C-terminal g domain is responsible for recognizing DNA sequences called FtsK recognition sequences (FRS). Single molecule experiments showed that deletion of the g domain eliminated FtsK's ability to change direction in response to FRS orientation, but did not affect its motor function or translocation speed. The researchers conclude the g domain allows FtsK to use FRS polarity to efficiently transport DNA by reducing nonproductive translocations after stochastic motor reversals.
This document describes a new microscopy technique called SPRAIPAINT that enables three-dimensional superresolution imaging of intracellular protein structures and the cell surface in living bacteria. SPRAIPAINT uses single molecule localization microscopy to image enhanced yellow fluorescent protein (eYFP) fusions within the cell and a dye binding to the cell surface sequentially using a single laser. This allows colocalization of an intracellular cytoskeletal protein (Crescentin-eYFP) with the cell membrane in living Caulobacter crescentus bacteria with 20-40nm precision over a large field of view. Imaging is done using a double-helix point spread function microscope for three-dimensional localization. SPRAIPAINT provides a
The document summarizes experiments studying the movement and directionality of the FtsK translocase protein on DNA molecules. Key findings include:
1) FtsK moves rapidly at 5 kilobases per second and can work against forces up to 60 piconewtons.
2) FtsK forms loops of DNA as it translocates and can reverse direction without dissociating from DNA.
3) When bound to lambda DNA, FtsK consistently moved in the same direction, toward the terminus region as predicted, even when the DNA was inverted.
This document summarizes recent research on the role of chromosome architecture in bacterial cellular organization. It discusses how chromosome structure encodes spatial information that directs processes like chromosome compaction, replication initiation, and segregation. Specifically, it describes how in Caulobacter, the positioning of the origin region near the cell pole facilitates cell cycle regulation. It also explains how the nucleoid provides a scaffold for partitioning systems to segregate chromosomal loci and other cargos through the cell. Finally, it discusses mechanisms in several bacteria by which dynamic changes to chromosome architecture couple replication and segregation to proper placement of the cell division machinery.
This document summarizes research on the chromosome segregation mechanism in the bacterium Caulobacter crescentus. The key points are:
1) C. crescentus was found to use a DNA partitioning (Par) system, involving the proteins ParA and ParB, to segregate its chromosome in a way similar to eukaryotic mitotic spindles.
2) Time-lapse microscopy showed ParA forms a retracting structure that leads the ParB-bound centromere (parS DNA) to the opposite cell pole during segregation. Super-resolution microscopy revealed ParA assembles into narrow linear structures in vivo.
3) In vitro experiments showed purified ParA polymer
1. The study demonstrates quantitative 3D super-resolution imaging of two fluorescent protein fusions in living bacterial cells using a dual-channel double-helix point spread function microscope.
2. Surface-relief phase masks are used to generate the double-helix PSF and allow precise 3D localization of single fluorescent molecules from two spectrally distinct labels over a large depth of field.
3. The positions of single molecules of photoactivatable mCherry and blinking eYFP fusions to two bacterial proteins are determined with nanometer precision and combined to reveal the 3D architecture of the labeled structures.
1) The document summarizes recent research on the mechanism of ParA-mediated chromosome segregation in bacteria.
2) Key findings include that ParA forms a structure along the bacterial nucleoid that is involved in segregating specific chromosomal loci.
3) A recent study found that in Caulobacter crescentus, ParA forms a narrow structure along the long axis of the cell, and the ParB/parS complex follows the edge of a receding ParA structure, providing evidence this is the mechanism of segregation.
1) The bacterial protein PopZ forms a scaffold at the cell pole that interacts directly with ParA to recruit it from the cytoplasm.
2) Mutations in PopZ that disrupt its interaction with ParA or ParB were identified, demonstrating PopZ interacts with both proteins.
3) PopZ recruits ParA released during chromosome segregation and sequesters it at the pole, stimulating reassembly of ParA on the nucleoid near the pole to drive continued segregation of chromosomes towards the pole. PopZ thus acts as a hub to spatially regulate chromosome segregation.
The mating-specific G protein (Gpa1) interacts with the kinesin motor protein Kar3 and regulates pheromone-induced nuclear migration in yeast. Upon pheromone stimulation, Gpa1 polarizes to the incipient growth site early in the mating response, around the same time that microtubules begin attaching to the cortex there. Gpa1 interacts with Kar3 in a microtubule-independent manner upon pheromone stimulation. In the absence of Gpa1, microtubules lose cortical contact upon shrinking and Kar3 is improperly localized, suggesting Gpa1 anchors Kar3 to the cortex. The authors infer that Gpa1 serves as a positional determinant for Kar3-bound microtub
This document provides evidence that common machinery is utilized by the early and late RNA localization pathways in Xenopus oocytes. It presents four key findings: 1) Early and late pathway RNAs require the same short sequence motifs for localization. 2) Competition assays show early and late RNAs compete for common localization factors in vivo. 3) A late localization factor, Vg RBP/Vera, binds specifically to localization elements of early pathway RNAs. 4) Confocal imaging reveals early RNAs associate with microtubules, suggesting transport plays a role in both pathways. Together, these findings suggest the early and late pathways share basal localization factors throughout oogenesis.
This document summarizes a study that found the transcription factors eyes absent (eya) and sine oculis (so), which are involved in eye development, are also required in the somatic cyst cells of the Drosophila testis for proper spermatocyte development. The study found that eya mutant testes exhibit degenerating young spermatocytes, and mosaic analysis revealed a somatic requirement for both eya and so in the cyst cells, not in the germline. Immunolocalization showed eya and so proteins are expressed in cyst cell nuclei as spermatocytes differentiate. The study suggests eya and so may form a transcription complex in the cyst cells to activate genes involved in cyst cell differentiation and s
The document discusses various aspects of genome organization, including:
1. Chromatin assembly begins with the incorporation of histone proteins to form nucleosomes, which are then folded and organized into higher order structures within the nucleus.
2. Genes can be split, overlapping, or pseudogenes. Split genes contain introns that are spliced out, while overlapping genes share nucleotide sequences. Pseudogenes are non-functional copies of genes.
3. Gene families consist of genes related by common ancestry that may be clustered or dispersed throughout the genome. Members can vary in sequence but often retain similar functions.
Introduction to genetics and genes unlocking the secrets of heredity by noor ...Noor Zada
Genetics is the study of heredity and the transmission of traits from parents to offspring. It examines inheritance at multiple levels, from whole organisms to chromosomes to genes and DNA. DNA is the genetic material found in all living things. It exists as long double-stranded helix molecules. DNA is made up of nucleotides containing phosphate, deoxyribose sugar, and nitrogenous bases. The bases pair up in a specific way between strands through hydrogen bonding to form the DNA code. DNA codes for traits by determining the sequence of proteins and RNA molecules. Its double helix structure allows for accurate copying and transmission of the genetic code during cell division.
This document summarizes a paper on self-renewal and differentiation of mouse embryonic stem cells. It discusses key transcription factors like Oct4, Nanog, Sox2, and Foxd3 that maintain the pluripotent and self-renewing properties of embryonic stem cells. The paper also describes how embryonic stem cells can be differentiated into various cell types through methods like embryoid body formation and growth factor exposure. Recent advances in using RNA interference and mutagenesis in embryonic stem cells are helping to better understand gene function in early development.
RNA localization to the Balbiani body in Xenopus oocytes is regulated by the energy state of the cell and is facilitated by kinesin II. The rate of RNA accumulation in the Balbiani body depends on temperature and intracellular ATP concentration - increasing ATP concentration doubles the localization rate. Inhibition of kinesin II reduces RNA localization to the Balbiani body, and the Xcat-2 RNA recruits kinesin II, indicating it plays a role in this process. The energy state of the cell regulates the rate of RNA transport to the Balbiani body, which involves kinesin II to some extent.
This document summarizes bacterial genetics and the structure and replication of bacterial chromosomes. It discusses how the bacterial chromosome is a circular DNA molecule that replicates semi-conservatively. The chromosome contains all the genetic information to determine the cell's characteristics. This information is expressed through transcription and translation. The document also covers genetic variation in bacteria through mutation, transformation, transduction, and conjugation which can result in new phenotypes and acquisition of new genes.
This document discusses the organization and segregation of bacterial chromosomes using Escherichia coli as an example. It finds that: (1) two markers on the same chromosome arm colocalize in the same cell half, while markers on opposite arms are in opposite cell halves, (2) duplicated chromosome arms are usually oriented in a tandem repeat configuration, and (3) sister cells are not usually identical after cell division due to rearrangement of chromosome arms.
Drosophila Melanogaster Genome And its developmental processSubhradeep sarkar
The document summarizes key aspects of the Drosophila genome and life cycle. It notes that Drosophila has advantages for genetic studies like a short life cycle and small genome. Its genome contains around 13,600 genes located on four chromosomes. The life cycle involves an egg, larva, pupa and adult stages. Segmentation and homeotic genes play important roles in development by dividing the body into segments and specifying segment identities. Maternal effect, gap, pair-rule and segment polarity genes control segmentation in a hierarchical manner. Homeotic complexes like bithorax determine body part identities in each segment.
4. 2 Intracellular Binding Partners Of Podocalyxin Lab StudyStephanie Roberts
This lab report examines cell motility and the role of cilia. Cilia are hair-like structures on cell surfaces that are important for cell movement and processing external signals during development. Approximately 600 cilia proteins must be synthesized inside cells and transported to cilia through intraflagellar transport. Disruptions to this transport can cause errors in cilia assembly. The report explores different types of cell movement and how the cytoskeleton, composed of microfilaments, microtubules, and intermediate filaments, is the main component enabling motility.
1. The document describes the development of a new single-cell RNA-seq method called Quartz-Seq that has higher reproducibility and sensitivity than existing methods.
2. Quartz-Seq can quantitatively detect various types of non-genetic cellular heterogeneity and can distinguish different cell types and cell cycle phases of a single cell type.
3. It can also comprehensively reveal gene expression heterogeneity between single cells of the same cell type in the same cell cycle phase.
This document summarizes bacterial genetics and the structure and replication of bacterial chromosomes. It notes that the bacterial chromosome is a single circular DNA molecule that replicates semi-conservatively. Genetic variation arises through mutation, horizontal gene transfer between bacteria, and extrachromosomal plasmids that can confer new traits. The genetic information is expressed through transcription and translation of DNA into mRNA and proteins.
This document discusses exon shuffling, which is a mechanism by which new genes can form through the rearrangement of exons from different genes. Exon shuffling was first proposed in 1978 and involves recombination within introns that allows exons to be assorted independently, generating new exon combinations. There are three main types of exon shuffling: exon duplication, insertion, and deletion. Exon shuffling generates genetic variation and mosaic proteins, and it has played a major role in evolution. The mechanisms involved are crossover during sexual recombination and transposon-mediated movements that can cut, paste, or copy and paste exons into new locations.
1) Bacterial cell division involves coordinating the segregation of replicated DNA and division of cytoplasmic material to generate progeny with identical genetic material. This requires spatial and temporal coordination of events.
2) The divisome, consisting of 10-15 proteins including FtsZ, assembles at midcell after DNA replication begins to assist in DNA separation and synthesize a new cell wall to divide the daughter cells.
3) There are multiple mechanisms that regulate divisome assembly to prevent it from occurring too early or in the same location as segregating chromosomes.
The document describes developmental patterns in several animal phyla. It discusses:
1) Spiral cleavage in mollusks and annelids, where cleavage planes are oblique, forming a spiral arrangement. This results in cells touching at more points than radially cleaving embryos.
2) Developmental axes in C. elegans defined by the anterior-posterior axis of the egg. The sperm entry point determines the posterior pole.
3) Radial holoblastic cleavage in sea urchins, where the first three cleavages are perpendicular, forming cell tiers, while the fourth cleavage divides cells meridionally.
In this PPT I completed that interesting topic , molecular embryology discussing this time molecular regulation of some other systems in the developing embryo, wishing that I could make this as simple as possible.
In this PPT I completed that interesting topic In this PPT I completed that interesting topic , molecular embryology discussing this time molecular regulation of some other systems in the developing embryo, wishing that I could make this as simple as possible.
cell lineage , cell fate - diverse class of cell fate, cell fate in plant meristem, mammalian development cell fate, nutritional effects on epigenetics, epigenetics of plants,
control of cell fate.
- The document discusses the establishment of polarity in the C. elegans zygote through the localization of proteins like PAR proteins, MEX-5, and PIE-1. PAR proteins first localize asymmetrically and then MEX-5 forms an anterior gradient dependent on PAR-1, while PIE-1 forms a posterior gradient. These gradients determine the fate of daughter cells after the first division.
2. Upon the initiation of sporulation, the replicated chro-
mosomes are remodeled into an elongated structure that
extends from one cell pole to the other. This serpentine
structure is called the axial filament (Ryter et al. 1966;
Ben-Yehuda et al. 2003b). The origin regions are located
at the extreme cell poles of the axial filament while the
termini reside at midcell (Lin et al. 1997; Webb et al.
1997; Wu and Errington 1998). As a result of axial fila-
ment formation, the asymmetric division plane traps ap-
proximately one quarter of the forespore chromosome
(the origin-proximal region) in the small forespore com-
partment (Wu and Errington 1998; N. Sullivan and D.Z.
Rudner, unpubl.). After the completion of cytokinesis,
SpoIIIE assembles on the DNA trapped in the septum
(Sharp and Pogliano 1999; Ben-Yehuda et al. 2003a) and
is responsible for pumping the remaining three quarters
of the chromosome (>3 megabases) from the mother cell
into the forespore (Wu and Errington 1994). Thus, in this
unusual cell cycle cytokinesis precedes DNA segrega-
tion.
An outstanding question related to the FtsK/SpoIIIE
family of transporters is what happens to the proteins
associated with the DNA during chromosome transloca-
tion (Fig. 1A). Are these proteins transported along with
the DNA across the division septum? Or are they
stripped off during translocation? In the case of sporula-
tion, either answer could have profound implications for
developmental gene expression. Since DNA-binding pro-
teins are predominantly associated with the chromo-
some and not free in the cytoplasm (Fig. 1B), if these
proteins are translocated along with the DNA, their con-
centration in the small forespore compartment could in-
crease significantly. Conversely, if these proteins are
stripped off the DNA during translocation, this could
help direct developmental gene expression by resetting
epigenetic nucleoprotein structures and/or by excluding
vegetative transcription factors from the developing
spore.
No study has addressed what happens to proteins as-
sociated with the chromosome during DNA transport
across the division septum. The pore size of the FtsK
hexameric ring in the crystal structure is ∼30 Å. Double-
stranded DNA could easily be threaded through this an-
nulus, but proteins associated with the DNA might not
be accommodated. In vitro experiments with the cyto-
plasmic motor domains of FtsK and SpoIIIE indicate that
these enzymes can displace ssDNA when it encounters a
DNA triplex (Bigot et al. 2005; Levy et al. 2005; Ptacin et
al. 2008), suggesting that DNA-binding proteins might
also be stripped off. However, some DNA-binding pro-
teins have very high affinity for their cognate binding
sites. In fact, in some instances these interactions are
sufficient to impede the movement of a translocating
complex as strong as the replisome (Possoz et al. 2006).
The biophysical data presently available for either FtsK
or SpoIIIE do not allow us to estimate whether these
enzymes could displace proteins tightly bound to DNA.
On the other hand, studies of several other AAA+
ATPases have revealed surprisingly elastic substrate ac-
commodation. Hexamer to heptamer transitions result-
ing in larger pore sizes have been described for bacterio-
phage T7 primase (Crampton et al. 2006). Moreover, re-
cent experiments analyzing protein translocation into
the AAA+ ClpXP proteosome revealed that this caged
protease with an annular entry port can translocate and
degrade branched substrates (Bolon et al. 2004). Thus,
Figure 1. Two models for the fate of DNA-binding proteins
during chromosome translocation. (A) In the first model DNA-
associated proteins (colored circles) are stripped off the forespore
chromosome during DNA transport. In the second model these
proteins are translocated into the forespore along with the
DNA. The SpoIIIE translocase (pink rectangle) is shown. The
origins of replication are located near the cell poles and the
termini are close to midcell. (B) RNA polymerase colocalizes
with the chromosome (nucleoid) inside the cell. Fluorescent
micrographs of vegetatively growing B. subtilis cells harboring
a functional Ј-GFP fusion (strain BDR1499). Ј-GFP colocalizes
with the DAPI-stained nucleoid. For comparison, a strain
(BDR1778) producing free GFP localizes as a diffuse haze
throughout the cytoplasm. A similar nucleoid-associated local-
ization was observed for Hbsu-GFP (data not shown). Higher
resolution versions of the figures are available in Supplemental
Material.
SpoIIIE strips proteins off DNA
GENES & DEVELOPMENT 1787
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
3. the channels of these translocation machines might be
more flexible than their crystal structures suggest.
Taking advantage of unique features of B. subtilis
sporulation, we analyzed the fate of proteins associated
with the DNA during chromosome translocation in
vivo. We demonstrate that RNA polymerase, chromo-
some remodeling proteins, and transcription factors are
stripped off the chromosome during DNA transport. In
particular, we show that fluorescent repressor proteins
(TetR-GFP) bound to an array of operators (tetO) are ef-
ficiently removed from the forespore chromosome dur-
ing translocation. By contrast, TetR-GFP bound to a tetO
array is sufficient to block progression of the replisome
during chromosome replication. These results are con-
sistent with the observation that the FtsK/SpoIIIE trans-
locase is a more powerful motor than the replisome. In
vitro experiments using the soluble motor domain of
SpoIIIE indicate that SpoIIIE can displace RNA polymer-
ase from transcription initiation complexes and stalled
elongation complexes. Finally, we present evidence that
the “wire-stripping” activity of SpoIIIE plays a role in
reprogramming developmental gene expression in the
forespore during sporulation.
Results
Proteins are stripped off the chromosome
during translocation in a disporic mutant
To investigate the fate of DNA-binding proteins during
chromosome translocation, we took advantage of a
sporulation mutant that forms septa at both poles of the
cell. These abortively disporic cells generate two fore-
spores separated by a mother cell compartment (Fig. 2A;
Stragier and Losick 1996). SpoIIIE assembles at both po-
lar septa, and the replicated chromosomes are pumped
into the two forespores, leaving the mother cell devoid of
DNA (Wu and Errington 1994). Since DNA-binding pro-
teins are predominantly associated with the chromo-
some (Figs. 1B, 2B), if these proteins are translocated
along with the DNA, the mother cell should be depleted
of DNA-binding proteins. On the other hand, if proteins
associated with the DNA are stripped off, then the pro-
teins initially bound to the chromosome in the mother
cell should be released into the mother cell cytoplasm
during DNA transport.
For these experiments we used a functional fusion of
the Ј subunit of RNA polymerase to GFP (rpoC-gfp). As
we observed for vegetative cells (Fig. 1B), RNA polymer-
ase specifically colocalized with the replicated chromo-
somes during sporulation in wild-type cells (Fig. 2B). In
the disporic mutant, when DNA translocation was not
yet complete, RNA polymerase colocalized with the
DNA in both the mother cell and forespore compart-
ments (Fig. 2B, yellow caret). However, in those cells in
which chromosome translocation was complete, as
judged by loss of DAPI signal (Fig. 2B, white carets), RNA
polymerase was observed as a diffuse haze in the mother
cell. Comparison of total Ј-GFP fluorescence intensity
in the mother cell before and after DNA translocation
indicates that 85%–90% of the RNA polymerase ini-
tially present in the mother cell was retained in the
mother cell compartment. Since proteins are unable to
diffuse between the two compartments (Wu and Erring-
ton 1997), we interpret these results to indicate that
Figure 2. RNA polymerase is stripped off the DNA during
chromosome translocation. (A) Schematic diagram of the ex-
periment in B with the two possible outcomes. An abortively
disporic mutant is shown with RNA polymerase (green balls)
associated with the replicated chromosomes. If RNA polymer-
ase is stripped off the DNA, the fluorescent fusion protein will
be present as a diffuse haze in the mother cell cytoplasm. If
RNA polymersase is translocated along with the DNA, the
mother cell will be devoid of fluorescent signal. The SpoIIIE
translocase (pink square) is shown. (B) Ј-GFP is stripped off the
chromosome during translocation into the forespore. Fluores-
cent micrographs of sporulating B. subtilis cells harboring a
functional Ј-GFP fusion. The top panels show the colocaliza-
tion of Ј-GFP with the nucleoid in wild-type sporulating cells
(strain BDR1499). The bottom panels show Ј-GFP in a disporic
mutant (strain BDR1522). An abortively disporic sporangium
that has not yet completed transport of the chromosomes into
the forespore compartments is shown (yellow caret). In this cell
Ј-GFP colocalizes with the DNA that is still present in the
mother cell compartment. Two abortively disporic sporangia
that have finished DNA translocation are indicated (white car-
ets). In these cells there is no DAPI signal in the mother cell and
Ј-GFP is visible as a diffuse haze in this compartment. The
signal appears weaker because the protein is no longer concen-
trated on the DNA. Quantitation of fluorescent intensity re-
vealed that 85%–90% of the mother cell signal remains after
DNA transport. Similar results were obtained with Hbsu-GFP
and RacA-GFP (data not shown). Bar, 1 µm.
Marquis et al.
1788 GENES & DEVELOPMENT
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
4. RNA polymerase was stripped off the chromosomes dur-
ing transport. The gene fusion was inserted at its endog-
enous locus (11° relative to the origin of replication), and
this region is trapped in the forespore compartment at
the time of polar septation. Thus, new transcription of
rpoC-gfp could only occur in the forespores during DNA
translocation. In fact, the signal in the forespores ap-
peared brighter at late time points (data not shown). We
also investigated the fate of two additional DNA-binding
proteins during chromosome translocation: the develop-
mentally regulated chromosome remodeling protein
RacA (RacA-GFP) (Ben-Yehuda et al. 2003b) and the
small histone-like protein Hbs (Hbs-GFP) (Kohler and
Marahiel 1997). In both cases a diffuse fluorescent signal
similar to Ј-GFP was observed in the mother cell com-
partment when DNA transport was complete (data not
shown). Altogether, these data are consistent with the
idea that DNA-binding proteins are stripped off the DNA
during chromosome translocation.
TetR-GFP is stripped off tetO arrays
during translocation
In the experiments described above, we visualized DNA-
binding proteins bound specifically and nonspecifically
throughout the genome. To investigate the fate of a tran-
scription factor bound site specifically, we used the TetR
repressor protein fused to GFP (TetR-GFP) and an array
of tet operators (tetO) (Michaelis et al. 1997). This re-
pressor-operator system has been used extensively to vi-
sualize the subcellular location of specific regions of the
chromosome in bacteria and eukaryotic cells (Michaelis
et al. 1997; Webb et al. 1997; Viollier et al. 2004; Wang et
al. 2005). When the tetO array was placed near the origin
of replication and the TetR-GFP fusion was synthesized
prior to polar division, two foci (one for each chromo-
some) could be easily visualized (Supplemental Fig. S1B).
As was found previously (Webb et al. 1997), one of the
foci was present in the mother cell and the other in the
forespore (Supplemental Fig. S1B).
To investigate the fate of TetR-GFP bound to the tetO
array during chromosome translocation, we placed the
tetR-gfp fusion under the control of a mother cell-spe-
cific promoter (PspoIID) such that the fluorescent repres-
sor protein would be synthesized exclusively in the
mother cell compartment (Fig. 3A). In addition, the tetO
array was inserted at a site (130°) that is present in the
mother cell at the time of polar septation and must be
translocated into the forespore (Fig. 3A). Accordingly, at
early time points after asymmetric division, two foci
(one for each chromosome) should be present in the
mother cell compartment. We could then assess the fate
of the TetR-GFP focus associated with the forespore
chromosome at late time points once DNA translocation
was complete (Fig. 3A). Unfortunately, using this experi-
mental setup we could only detect one fluorescent focus
in the mother cell (and none in the forespore) at early
time points (data not shown). Whereas the SpoIIIE ini-
tiates DNA transport immediately after division is com-
plete, the first mother cell-specific transcription factor
(E
) is activated only after a short delay (Zhang et al.
1996; Zupancic et al. 2001). Thus, we suspect that the
terminus region of the chromosome (including the tetO
array) is transported into the forespore before TetR-GFP
accumulates (or folds or matures) to significant levels.
To circumvent this problem, we took advantage of a
newly discovered SpoIIIE mutant (SpoIIIED584A
) whose
rate of DNA transport is reduced by 2.5-fold (Burton et
al. 2007). Using this mutant background, we could detect
two TetR-GFP foci in the mother cell in most (79%,
n = 297) of the sporulating cells at early time points (Fig.
3B). Consistent with the idea that TetR-GFP is stripped
off the DNA, at late time points, when chromosome trans-
port was complete, we could detect a single bright focus
in the mother cell and no focus in the forespore in vir-
tually all (98%, n = 324) of the cells (Fig. 3B). Similar
results were obtained when tetO arrays were placed at
100° or near the terminus at 162° (data not shown). As re-
ported previously, arrays close to the terminus are not
easily resolved prior to translocation (Bogush et al. 2007).
However, moments before translocation of the 162° re-
gion, we could detect two foci in the mother cell com-
partment of some cells (Supplemental Fig. S2). Impor-
tantly, we never observed foci in the forespores. Alto-
gether, these results indicate that DNA-binding proteins
are stripped off the DNA during chromosome translocation.
Similar results were obtained in a strain containing
wild-type SpoIIIE. In this strain TetR-GFP was synthe-
sized at the onset of sporulation in the predivisional cell
(using the PspoIIE promoter), and upon polar septation its
synthesis was shut down in the forespore but persisted in
the mother cell compartment (Supplemental Fig. S1A;
Fujita and Losick 2003). In this background, when the
tetO array was present at −91°, two bright foci could be
seen in the mother cell upon polar division (Supplemen-
tal Fig. S1B). Once translocation was complete, a bright
focus was visible in the mother cell and a faint focus
could be detected in the forespore. We interpret these
results to mean that the mother cell TetR-GFP was
stripped off during DNA translocation and the low levels
of TetR-GFP present in the forespore bound these sites
upon their arrival in the forespore compartment. Consis-
tent with this idea, when the tetO array was placed near
the origin (−7°), such that at the time of polar division
the array was already present in the forespore compart-
ment, the mother cell and forespore foci remained at
equal intensity after DNA translocation (Supplemental
Fig. S1B).
Repressors bound to arrays of operators have been
found to block the progression of the replisome in Esch-
erichia coli (Possoz et al. 2006). We observed a similar
replication roadblock in B. subtilis (Supplemental Fig.
S4). In the experiments described above TetR-GFP was
synthesized after completion of DNA replication but
prior to DNA transport. We wondered whether TetR-
GFP bound to the tetO array might impede or slow down
DNA translocation and therefore cause a sporulation de-
fect. To test this, we analyzed sporulation efficiency in
the strains described above. In support of the idea that
SpoIIIE efficiently strips these proteins off the DNA, in
SpoIIIE strips proteins off DNA
GENES & DEVELOPMENT 1789
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
5. all strains tested the sporulation efficiency was similar
to wild type (Supplemental Table S1). These results are
consistent with reports that FtsK/SpoIIIE translocases
are stronger motors than the replisome (Wuite et al.
2000; Pease et al. 2005) and altogether these experiments
support the view that the forespore chromosome arrives
naked in the developing spore.
The ATPase domain of SpoIIIE can displace RNA
polymerase bound to DNA in vitro
To investigate whether SpoIIIE is responsible for clearing
proteins from the DNA during translocation, we ana-
lyzed the ability of SpoIIIE to displace RNA polymerase
bound to DNA in vitro. For these experiments we used
the soluble motor domain of SpoIIIE, which has been
shown to translocate along DNA in vitro (Bath et al.
2000; Ptacin et al. 2008). E. coli RNA polymerase con-
taining 70
was assembled on a fluorescently labeled
DNA substrate containing the PR promoter. Complexes
were formed in the absence of ribonucleotides to gener-
ate an initiation complex or in the presence of three of
the four ribonucleotides to create a stalled elongation
complex (see the Materials and Methods). Both com-
plexes were stable in a gel mobility shift assay (Fig. 4;
Supplemental Fig. S3). When these complexes were in-
cubated with either the motor domain of SpoIIIE or high
concentrations of ATP, RNA polymerase remained sta-
bly associated with the DNA (Fig. 4A; Supplemental Fig.
S3). However, when both SpoIIIE and ATP were present,
RNA polymerase was displaced from the DNA substrate
(Fig. 4A; Supplemental Fig. S3). To confirm that the dis-
placed complex was a stalled elongation complex, we
followed the fate of the RNA transcript by generating
stalled complexes as described above but in the presence
of ␣32
P-GTP (see the Materials and Methods). This ter-
nary complex composed of RNA polymerase, a radioac-
tive transcript, and the substrate DNA was stable in a gel
mobility shift assay and remained intact in the presence
of either the motor domain of SpoIIIE or high concentra-
tions of ATP (Fig. 4B). Addition of both SpoIIIE and ATP
efficiently dissociated the ternary complex (Fig. 4B). We
interpret this to mean that the ATP-dependent motor
Figure 3. Transcriptional regulators are stripped off the DNA
during translocation. (A) Schematic diagram of the experiment
in B with the two possible outcomes. An array of tetO operators
was inserted at a position near the teriminus (130°) that is al-
ways present in the mother cell at the time of polar septation. In
this strain, the tetR-gfp fusion was placed under the control of
a mother cell-specific (E
-dependent) promoter. Finally, this
strain has a mutant SpoIIIE translocase (pink square) that pumps
the chromosome 2.5-fold slower than wild type (Burton et al.
2007). Thus, at early time points after E
becomes active in the
mother cell, two TetR-GFP foci should be visible in the mother
cell. If TetR-GFP is stripped off the DNA, one of these two foci
will be missing when chromosome translocation is complete. If
TetR-GFP is translocated with the chromosome, when DNA
transport is complete, one TetR-GFP focus will be visible in the
forespore and one in the mother cell. (B) TetR-GFP bound to the
tetO array is stripped off the chromosome during translocation.
Fluorescence micrographs of sporulating B. subtilis cells (strain
BKM1016) at early (hour 2) and late (hour 3) time points. At hour
2, prior to the completion of DNA translocation, two fluores-
cent foci are visible in the mother cell (yellow carets). At hour
3, when chromosome translocation is complete in most cells, a
single focus is visible in the mother cell (yellow caret) and no
detectable foci are seen in the forespore (white caret). No TetR-
GFP foci are visible in the forespore when the micrograph is
rescaled (overexposed) to reveal weak signals. Bar, 1 µm.
Marquis et al.
1790 GENES & DEVELOPMENT
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
6. activity of SpoIIIE stripped RNA polymerase from the
DNA, resulting in release of the labeled transcript (An-
drews and Richardson 1985). Taken together, these re-
sults indicate that the motor domain of SpoIIIE can dis-
place RNA polymerase during transcription initiation
and in the context of a stalled elongation complex. Dis-
sociation of transcription elongation complexes has been
described previously for the E. coli transcription repair
coupling factor (Mfd) (Selby and Sancar 1993). Although
it is possible that an analogous protein participates in
displacing RNA polymerase in vivo, our in vitro data
suggest that SpoIIIE on its own is capable of removing
these complexes during DNA transport.
Vegetative transcription is down-regulated
in the forespore
We wondered whether stripping of RNA polymerase and
its associated vegetative (housekeeping) factor (A
)
might result in lower transcription from A
-dependent
promoters in the forespore compared to the mother cell.
Attempts to monitor expression of vegetative promoters
fused to gfp during sporulation were not informative due
to the relative stability of GFP and its production during
vegetative growth prior to polar division (data not
shown). To circumvent these problems, we fused the
fluorescent reporter to a xylose-inducible A
-dependent
promoter (PxylA) (Fig. 5A). Accordingly, we could induce
A
-dependent gene expression during chromosome
translocation and monitor GFP levels shortly after its
completion. The PxylA-gfp reporter was inserted at a po-
sition (28°) in the chromosome that is trapped in the
forespore at the time of polar division (Fig. 5A). There-
fore, at the time of induction, both the mother cell and
forespore contain a copy of the reporter. Synchronous
sporulation was induced and, when ∼40% of the cells
had a polar septum (hour 1) (Fig. 5A) and were just be-
ginning DNA transport, xylose was added to the culture.
Thirty minutes later (hour 1.5) GFP was visualized by
fluorescence microscopy. In those cells that had justFigure 4. The soluble motor domain of SpoIIIE can displace a
stalled transcription elongation complex in vitro. (A) E. coli
RNA polymerase was incubated with a fluorescently labeled
DNA substrate containing the PR promoter. The reaction con-
tained 25 µM UTP, GTP, and ATP but no CTP to generate a
stalled elongation complex (EC). The stalled complexes were
then incubated with 3 mM ATP, 420 nM SpoIIIE, and 3 mM
ATP, 420 nM SpoIIIE, or 5% SDS and analyzed by gel mobility
shift on a native polyacrylamide gel. The fraction of RNA poly-
merase present in the elongation complex is indicated below
the gel. (B) E. coli RNA polymerase was incubated with the PR
promoter and 2.5 µM UTP, GTP, ATP and 0.7 µM ␣32
P-GTP but
no CTP to generate a stalled elongation complex with a short
radiolabeled transcript. The stalled complex was incubated with
3 mM ATP, 200 nM SpoIIIE, and 3 mM ATP, 200 nM SpoIIIE, or
5% SDS and the reactions were analzyed on a native polyacryl-
amide gel followed by autoradiography. Dissassembly of the
complex is evidenced by release of the labeled transcript. The
stalled complex was confirmed by the ability of NspI to cut the
DNA substrate (+NspI) within the promoter region, resulting in
a faster migrating complex.
Figure 5. Vegetative gene expression is down-regulated in the
forespore after chromosome translocation. As a result of the
wire-stripping activity of SpoIIIE, much of A
-associated RNA
polymerase will be stripped off the forespore chromosome dur-
ing translocation. One prediction of this model is that A
-di-
rected transcription will be reduced in the forespore compared
to the mother cell. (A) The xylose-inducible A
promoter PxylA
was fused to gfp and placed at an original-proximal position of
the chromosome (strain BDR734), and synchronous sporulation
was induced in the absence of xylose. When ∼40% of the sporu-
lating cells had generated a polar septum (hour 1) xylose was
added (20 mM final concentration) to induce transcription from
the PxylA promoter. (B) GFP fluoresence was visualized 30 min
later (hour 1.5). In sporulating cells in which DNA translocation
had initiated or was complete, GFP fluorescence was reduced in
the forespore (white caret) compared to the mother cell (yellow
caret). In cells that had just assembled an asymmetric septum,
the signal in both compartments was similar (red caret). Cell-
to-cell variability in the intensity of the GFP signal is due to the
differences in stages of sporulation at the time of xylose addi-
tion. Bar, 1 µm.
SpoIIIE strips proteins off DNA
GENES & DEVELOPMENT 1791
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
7. formed a polar septum, the GFP signal was similar in
both the mother cell and forespore compartments (red
caret in Fig. 5B). However, in cells that had been trans-
locating their DNA during the induction period, there
was less GFP signal in the forespore than the mother cell
(yellow and white carets in Fig. 5B). Li and Piggot (2001)
have shown previously that A
continues to be active in
the forespore both before and after completion of engulf-
ment. Consequently, some A
activity in the forespore
was expected. There was some cell-to-cell variability in
the intensity of GFP signal due to differences in the de-
velopmental stage of the sporulating cells at the time of
xylose addition (i.e., whether or not polar septation had
occurred). Accordingly, the relevant comparison is be-
tween mother cell and forespore in the same sporangia. It
is not known whether xylose continues to be transported
into the forespore once engulfment is complete. It is
therefore possible that the reduced expression from this
promoter could be due, in part, to lower concentrations
of the inducer in the spore compartment. However, clear
differences in the levels of GFP in the mother cell and
forespore could be observed at early stages of engulfment
when both cells were in direct contact with the medium
(Fig. 5B). These results suggest that A
-dependent tran-
scription is lower in the forespore than the mother cell
and are consistent with the idea that stripping A
from
the forespore chromosome facilitates the down-regula-
tion of housekeeping gene expression in the forespore
compartment.
Discussion
We show that RNA polymerase, a transcriptional regu-
lator, and chromosome packaging/remodeling proteins
are stripped off the chromosome during DNA transport.
Moreover, our in vitro analysis is consistent with the
idea that SpoIIIE alone is responsible for this wire-strip-
ping activity. If, as our data suggest, all proteins are
stripped off the forespore chromosome during DNA
transport, then ∼75% of the forespore genome (>3 Mb of
DNA) arrives in the forespore compartment naked ready
to receive and carry out the dictates of the developing
spore. We hypothesize that stripping plays a key role in
reprogramming developmental gene expression by reset-
ting epigenetic nucleoprotein structures and reducing
the concentration of vegetative transcription factors in
the developing spore.
Our observation that A
-dependent gene expression is
reduced in the forespore compartment compared to the
mother cell is consistent with the idea that stripping
down-regulates vegetative gene expression in the fore-
spore. The down-regulation of housekeeping genes could
allow the developing spore to focus its attention on the
preparation for dormancy. We note that our experiments
cannot distinguish between reduced A
-dependent gene
expression due to stripping of A
and the possibility that
the activation of F
in the forespore results in competi-
tion for core RNA polymerase (Ju et al. 1999; Grigorova
et al. 2006) and therefore a reduced A
holoenzyme pool.
However, these models are not mutually exclusive. F
is
made in the predivisional cell but is held inactive in the
cytoplasm by the anti-sigma factor SpoIIAB (Stragier and
Losick 1996; Rudner and Losick 2001; Hilbert and Piggot
2004). When cytokinesis is complete, F
is released from
SpoIIAB in the forespore compartment where it associ-
ates with core RNA polymerase. Stripping A
off the
forespore chromosome likely helps shift the balance in
favor of F
and thus helps lock the forespore into its
developmental program (Lord et al. 1999).
It is noteworthy that Fujita and Losick (2003) have
found that gene expression under the control of the tran-
scription factor Spo0A (the master regulator for entry
into the sporulation pathway) is similarly lower in the
forespore compared to the mother cell (Supplemental
Fig. S1A). Moreover, these researchers showed that inap-
propriate expression of Spo0A-controlled genes in the
forespore causes a 50-fold reduction in sporulation effi-
ciency. It is possible, and we think likely, that stripping
of Spo0A off the forespore chromosome during DNA
transport contributes to the down-regulation of Spo0A-
controlled genes in the forespore compartment. In sup-
port of this idea, localization of Spo0A by immuno-
fluorescence microscopy indicates that the levels of the
transcription factor are lower in the forespore than the
mother cell (Fujita and Losick 2003).
The removal of proteins from the forespore chromo-
some during transport likely plays an additional role in
maintaining compartment-specific gene expression. The
first mother cell-specific transcription factor E
becomes
active shortly after polar division and prior to the
completion of DNA transport (Zhang et al. 1996; Zupan-
cic et al. 2001). Thus, removal of E
associated with the
forespore chromosome would ensure that production of
mother cell proteins under the control of E
is restricted
to the mother cell compartment.
Another possible role for the stripping activity of
SpoIIIE is in chromosome remodeling. As part of the
preparation for dormancy, the forespore synthesizes a set
of small acid-soluble proteins that compacts the chro-
mosome into a doughnut-like structure (Pogliano et al.
1995) and confers resistance to DNA damaging agents
(Setlow 1995). The stripping of vegetative proteins off
the DNA could facilitate the remodeling of the chromo-
some into this dormant structure later in development.
Our data suggest that SpoIIIE can efficiently remove
RNA polymerase from the chromosome during DNA
transport. However, it is possible that head-on collisions
between SpoIIIE and RNA polymerase might affect the
rate of chromosome transport. Interestingly, the major-
ity (∼75%) of the genes in the B. subtilis genome are
transcribed toward the terminus and this unusually high
bias is found in all endospore formers (Rocha 2004). It
has been proposed that this coorientation with replisome
movement has been selected to avoid collisions between
RNA and DNA polymerases (Brewer 1988; French 1992).
We wonder whether DNA translocation and the conse-
quence of multiple head-on collisions between the tran-
scription machinery and SpoIIIE might serve as an added
selective pressure for coorientation.
In conclusion, we showed that DNA-binding proteins
Marquis et al.
1792 GENES & DEVELOPMENT
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
8. are actively removed from the chromosome during DNA
translocation. Our in vitro studies suggest that SpoIIIE is
sufficient for this wire-stripping activity; however, we do
not exclude the possibility that other proteins also par-
ticipate in removing tightly bound proteins during the
translocation process. Finally, our data and those of oth-
ers are consistent with the idea that the removal of
DNA-binding proteins plays an important role in dictat-
ing developmental gene expression in the forespore. It
remains unclear what impact, if any, the additional
DNA-binding proteins removed from the forespore chro-
mosome have on gene expression and chromosome dy-
namics in the mother cell. During vegetative growth, we
suspect that SpoIIIE (and other FtsK/SpoIIIE family
members) also removes proteins during the transport of
missegregated DNA. In E. coli two recombination pro-
teins, XerC and XerD, bind sequence elements (dif sites)
near the replication terminus. These factors interact
with FtsK, and this interaction is required for resolution
of chromosome dimers (Aussel et al. 2002). The displace-
ment of these proteins from the DNA prior to recombi-
nation would prevent dimer resolution. Thus, we hy-
pothesize that interaction between XerC/D and FtsK pre-
vents translocation and stripping until recombination is
complete.
Materials and methods
General methods
All B. subtilis strains were derived from the prototrophic strain
PY79 (Youngman et al. 1983) and are listed in Supplemental
Table S2. E. coli strains were TG1 and DH5␣. Sporulation was
induced by resuspension at 37°C according to the method of
Sterlini-Mandelstam (Harwood and Cutting 1990). Sporulation
efficiency was determined in 36 h cultures as the total number
of heat-resistant (80°C for 20 min) colony forming units (CFU)
compared with the number of wild-type heat-resistant CFU. To
prevent selection for shrunken tetO arrays, strains containing
(tetO)120 and tetR-gfp were maintained in the presence of 40
ng/mL anhydrotetracycline. The inducer was not present during
experiments. Plasmids and oligonucleotide primers used in this
study are listed in Supplemental Tables S3 and S4.
Fluorescence microscopy
Fluorescence microscopy was performed with an Olympus
BX61 microscope as described (Doan et al. 2005). Fluorescent
signals were visualized with a phase contrast objective Uplan-
FLN 100× and captured with a monochrome CoolSnapHQ digi-
tal camera (Photometrics) using Metamorph software version
6.1 (Universal Imaging). Exposure times were typically 500
msec for GFP fusions (excitation: 490–510 nm; emission: 520–
550 nm). Membranes were stained with either TMA-DPH (ex-
citation: 340–380 nm; emission: 435–485 nm) or FM4-64 (exci-
tation: 510–550 nm; emission: 590 nm longpass) with exposure
times of 200 msec. DNA was visualized with DAPI (excitation:
340–380 nm; emission: 435–485 nm) with exposure times of 200
msec. Images were analyzed, adjusted, and cropped using Meta-
morph software. Higher resolution images can be found in the
Supplemental Material.
RNA polymerase stalled elongation complex displacement
assays
DNA substrates for RNA polymerase displacement reactions
contained the PR promoter followed by a 70-bp-long stretch
devoid of guanosines in the template strand. Omission of CTP
led to the stalling of RNA polymerase at the position of the first
template guanosine.
To create the fluorescently labeled DNA substrates, a 433-bp
DNA fragment containing the PR promoter was PCR-amplified
from pJP101 using fluorescently labeled oligonucleotides
(oJP036 and oJP037). This PCR product was purified using the
Qiagen PCR-cleanup kit. Stalled complexes were prepared in
TBF Buffer (40 mM Tris-HCl at pH 7.5, 10 mM MgCl2, 0.01%
Triton X-100, and 1 mM DTT) by incubating 100 nM E.coli
RNA polymerase holoenzyme (Epicentre) and 20 nM fluores-
cent 433-bp PR DNA fragment for 30 min at 37°C. The reaction
was further incubated for 10 min at 22°C in the presence of 100
nM nonfluorescent 433-bp PR DNA to compete unbound and
nonspecifically bound RNA polymerase. To produce stalled
elongation complexes, a nucleotide mixture containing GTP,
UTP, and ATP at a final concentration of 25 µM was incubated
with the transcription reaction for 20 min at 22°C.
Displacement of stalled RNA polymerase by SpoIIIE was as-
sayed by incubating 10 nM stalled elongation complex with 420
nM of the SpoIIIE motor domain (SpoIIIEC) (Ptacin et al. 2008)
and 3 mM ATP in SpoIIIEC reaction buffer (50 mM Tris-HCl at
pH 7.5, 10 mM MgCl2). All reactions were incubated for 10 min
at 37°C and then stopped by incubation with 0.2 mg/mL hepa-
rin for 5 min at 22°C. Reactions were analyzed in 4%–20%
precast polyacrylamide gels (Bio-Rad) and run in 1× TBE (45 mM
Tris-borate at pH 8.3, 1 mM EDTA) at 20 mA for 2 h at 22°C.
Gels were imaged using a Typhoon scanner (GE) and bands
quantified using ImageQuant.
To create nonfluorescent stalled elongation complexes with
radiolabeled RNA transcripts, a 278-bp DNA fragment contain-
ing PR was PCR-amplified from pJP101 (oligonucleotide prim-
ers oJP033 and oJP034) and purified using the Qiagen PCR-
cleanup kit. Stalled complexes were prepared in TBF Buffer by
incubating 50 nM RNA polymerase and 100 nM DNA template
for 30 min at 37°C. To produce stalled elongation complexes,
the mixture was incubated with GTP, UTP, and ATP at final
concentrations of 2.5 µM, and 0.17 µM ␣32
P-GTP for 20 min at
22°C.
Displacement of radiolabeled complexes by SpoIIIE was as-
sayed by incubating 5 nM radioactive stalled elongation com-
plex with 200 nM SpoIIIEC, 3 mM ATP, and 25 µM unlabeled
GTP in SpoIIIEC reaction buffer for 10 min at 37°C. Unlabeled
GTP was added to the reaction to prevent the formation of new
radiolabeled stalled complexes resulting from the reassociation
of RNA polymerase with PR DNA after displacement. Reac-
tions treated with NspI were performed in 1× NEB2 buffer with
1 mg/mL BSA and 0.025 units of NspI per microliter. All reac-
tions were incubated for 10 min at 37°C and then stopped by
incubation with 0.2 mg/mL heparin for 5 min at 22°C. Gels
were run as described above, exposed on phosphorimager
screens (Amersham Biosciences), and imaged using a Typhoon
scanner.
Plasmid construction
pKM193 [pelBϻPspoIID-tetR-gfp (tet)] was generated in a three-
way ligation with a HindIII–BamHI PCR product containing
tetR-gfp and an optimized ribosome-binding site (Vellanoweth
and Rabinowitz 1992) (oligonucleotide primers oDR78 and
oDR188 with pBW3 [Dworkin and Losick 2002] as template)
SpoIIIE strips proteins off DNA
GENES & DEVELOPMENT 1793
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
9. and an EcoRI–HindIII PspoIID promoter fragment from pDR78
(Rudner and Losick 2002) and pKM033 (pelBϻtet) cut with
EcoRI and BamHI. pKM033 is an ectopic integration vector for
double cross-over integrations at the nonessential pelB locus
(K.A. Marquis and D.Z. Rudner, unpubl.).
pKM219 [ykpAB (130°) (tetO)120 (spec)] was generated by in-
serting a PCR product containing the (tetO)120 array (oligonu-
cleotide primers oDR458 and oDR459 with pLAU44 [Lau et al.
2003] as template) into pKM210 between HindIII and XhoI.
pKM210 was generated by inserting a 600-bp PCR product con-
taining part of ykpA and ykpB (oligonucleotide primers oDR494
and oDR495 with PY79 genomic DNA as template) into pUS19
(pUC19-spec) between EcoRI and BamHI. pKM210 is a single
cross-over integration vector for insertions at 130° (K.A. Mar-
quis and D.Z. Rudner, unpubl.).
pJP101 [PR promoter] was generated by inserting a PCR prod-
uct containing the PR promoter (oligonucleotide primer oJP033
and oJP034 with pPIA2-6 as template [Davenport et al. 2000])
into the SacII site of pJB103 (Bath et al. 2000).
Acknowledgments
We thank members of the Rudner laboratory past and present,
Tom Rapoport, Richard Losick, Antoine Van Oijen, and Ann
Hochschild for valuable discussions. We acknowledge Masaya
Fujita, Alan Grossman, and David Sherratt for strains and plas-
mids and Remi Bernhard for help with microscopy. This project
was initiated by D.Z.R. and S.B. when they were postdoctoral
fellows in the laboratory of R. Losick, and these authors wish to
acknowledge his enduring mentorship. This paper is dedicated
to Nick Cozzarelli. Support for this work comes from the Na-
tional Institute of Health Grant GM073831-01A1 and the Hell-
man Family Faculty Fund. D.Z.R. is supported by the Damon
Runyon Cancer Research Foundation (DRS 44-05). B.M.B was
supported in part by the Damon Runyon Cancer Research Foun-
dation (DRG 1804-04) and by the Charles A. King Trust, Bank of
America, Co-Trustee. M.N. was supported by the Human Fron-
tier Science Program.
References
Andrews, C. and Richardson, J.P. 1985. Transcription termina-
tion factor mediates simultaneous release of RNA tran-
scripts and DNA template from complexes with Escherichia
coli RNA polymerase. J. Biol. Chem. 260: 5826–5831.
Aussel, L., Barre, F.X., Aroyo, M., Stasiak, A., Stasiak, A.Z., and
Sherratt, D. 2002. FtsK is a DNA motor protein that acti-
vates chromosome dimer resolution by switching the cata-
lytic state of the XerC and XerD recombinases. Cell 108:
195–205.
Bath, J., Wu, L.J., Errington, J., and Wang, J.C. 2000. Role of
Bacillus subtilis SpoIIIE in DNA transport across the mother
cell-prespore division septum. Science 290: 995–997.
Ben-Yehuda, S., Rudner, D.Z., and Losick, R. 2003a. Assembly
of the SpoIIIE DNA translocase depends on chromosome
trapping in Bacillus subtilis. Curr. Biol. 13: 2196–2200.
Ben-Yehuda, S., Rudner, D.Z., and Losick, R. 2003b. RacA, a
bacterial protein that anchors chromosomes to the cell
poles. Science 299: 532–536.
Bigot, S., Saleh, O.A., Lesterlin, C., Pages, C., El Karoui, M.,
Dennis, C., Grigoriev, M., Allemand, J.F., Barre, F.X., and
Cornet, F. 2005. KOPS: DNA motifs that control E. coli chro-
mosome segregation by orienting the FtsK translocase.
EMBO J. 24: 3770–3780.
Bogush, M., Xenopoulos, P., and Piggot, P.J. 2007. Separation of
chromosome termini during sporulation of Bacillus subtilis
depends on SpoIIIE. J. Bacteriol. 189: 3564–3572.
Bolon, D.N., Grant, R.A., Baker, T.A., and Sauer, R.T. 2004.
Nucleotide-dependent substrate handoff from the SspB adap-
tor to the AAA+ ClpXP protease. Mol. Cell 16: 343–350.
Brewer, B.J. 1988. When polymerases collide: Replication and
the transcriptional organization of the E. coli chromosome.
Cell 53: 679–686.
Britton, R.A. and Grossman, A.D. 1999. Synthetic lethal phe-
notypes caused by mutations affecting chromosome parti-
tioning in Bacillus subtilis. J. Bacteriol. 181: 5860–5864.
Burton, B.M., Marquis, K.A., Sullivan, N.L., Rapoport, T.A., and
Rudner, D.Z. 2007. The ATPase SpoIIIE transports DNA
across fused septal membranes during sporulation in Bacil-
lus subtilis. Cell 131: 1301–1312.
Crampton, D.J., Ohi, M., Qimron, U., Walz, T., and Richardson,
C.C. 2006. Oligomeric states of bacteriophage T7 gene 4 pri-
mase/helicase. J. Mol. Biol. 360: 667–677.
Davenport, R.J., Wuite, G.J., Landick, R., and Bustamante, C.
2000. Single-molecule study of transcriptional pausing and
arrest by E. coli RNA polymerase. Science 287: 2497–2500.
Doan, T., Marquis, K.A., and Rudner, D.Z. 2005. Subcellular
localization of a sporulation membrane protein is achieved
through a network of interactions along and across the sep-
tum. Mol. Microbiol. 55: 1767–1781.
Dworkin, J. and Losick, R. 2002. Does RNA polymerase help
drive chromosome segregation in bacteria? Proc. Natl. Acad.
Sci. 99: 14089–14094.
Errington, J. 2003. Regulation of endospore formation in Bacil-
lus subtilis. Nat. Rev. Microbiol. 1: 117–126.
Errington, J., Bath, J., and Wu, L.J. 2001. DNA transport in bac-
teria. Nat. Rev. Mol. Cell Biol. 2: 538–545.
French, S. 1992. Consequences of replication fork movement
through transcription units in vivo. Science 258: 1362–1365.
Fujita, M. and Losick, R. 2003. The master regulator for entry
into sporulation in Bacillus subtilis becomes a cell-specific
transcription factor after asymmetric division. Genes & Dev.
17: 1166–1174.
Grigorova, I.L., Phleger, N.J., Mutalik, V.K., and Gross, C.A.
2006. Insights into transcriptional regulation and compe-
tition from an equilibrium model of RNA polymerase bind-
ing to DNA. Proc. Natl. Acad. Sci. 103: 5332–5337.
Harwood, C.R. and Cutting, S.M. 1990. Molecular biological
methods for Bacillus. Wiley, New York.
Hilbert, D.W. and Piggot, P.J. 2004. Compartmentalization of
gene expression during Bacillus subtilis spore formation. Mi-
crobiol. Mol. Biol. Rev. 68: 234–262.
Ju, J., Mitchell, T., Peters 3rd, H., and Haldenwang, W.G. 1999.
Factor displacement from RNA polymerase during Bacil-
lus subtilis sporulation. J. Bacteriol. 181: 4969–4977.
Kohler, P. and Marahiel, M.A. 1997. Association of the histone-
like protein HBsu with the nucleoid of Bacillus subtilis. J.
Bacteriol. 179: 2060–2064.
Lau, I.F., Filipe, S.R., Soballe, B., Okstad, O.A., Barre, F.X., and
Sherratt, D.J. 2003. Spatial and temporal organization of rep-
licating Escherichia coli chromosomes. Mol. Microbiol. 49:
731–743.
Levy, O., Ptacin, J.L., Pease, P.J., Gore, J., Eisen, M.B., Busta-
mante, C., and Cozzarelli, N.R. 2005. Identification of oli-
gonucleotide sequences that direct the movement of the
Escherichia coli FtsK translocase. Proc. Natl. Acad. Sci. 102:
17618–17623.
Li, Z. and Piggot, P.J. 2001. Development of a two-part tran-
scription probe to determine the completeness of temporal
and spatial compartmentalization of gene expression during
Marquis et al.
1794 GENES & DEVELOPMENT
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from
10. bacterial development. Proc. Natl. Acad. Sci. 98: 12538–
12543.
Lin, D.C., Levin, P.A., and Grossman, A.D. 1997. Bipolar local-
ization of a chromosome partition protein in Bacillus subti-
lis. Proc. Natl. Acad. Sci. 94: 4721–4726.
Lord, M., Barilla, D., and Yudkin, M.D. 1999. Replacement of
vegetative A by sporulation-specific F as a component of
the RNA polymerase holoenzyme in sporulating Bacillus
subtilis. J. Bacteriol. 181: 2346–2350.
Massey, T.H., Mercogliano, C.P., Yates, J., Sherratt, D.J., and
Lowe, J. 2006. Double-stranded DNA translocation: Struc-
ture and mechanism of hexameric FtsK. Mol. Cell 23: 457–
469.
Michaelis, C., Ciosk, R., and Nasmyth, K. 1997. Cohesins:
Chromosomal proteins that prevent premature separation of
sister chromatids. Cell 91: 35–45.
Pease, P.J., Levy, O., Cost, G.J., Gore, J., Ptacin, J.L., Sherratt, D.,
Bustamante, C., and Cozzarelli, N.R. 2005. Sequence-di-
rected DNA translocation by purified FtsK. Science 307:
586–590.
Pogliano, K., Harry, E., and Losick, R. 1995. Visualization of the
subcellular location of sporulation proteins in Bacillus sub-
tilis using immunofluorescence microscopy. Mol. Micro-
biol. 18: 459–470.
Possoz, C., Filipe, S.R., Grainge, I., and Sherratt, D.J. 2006.
Tracking of controlled Escherichia coli replication fork stall-
ing and restart at repressor-bound DNA in vivo. EMBO J. 25:
2596–2604.
Ptacin, J.L., Nollmann, M., Becker, E.C., Cozzarelli, N.R., Pogli-
ano, K., and Bustamante, C. 2008. Sequence-directed DNA
export guides chromosome translocation during sporulation
in Bacillus subtilis. Nat. Struct. Mol. Biol. 15: 485–493.
Rocha, E.P. 2004. The replication-related organization of bacte-
rial genomes. Microbiol. 150: 1609–1627.
Rudner, D.Z. and Losick, R. 2001. Morphological coupling in
development: Lessons from prokaryotes. Dev. Cell 1: 733–742.
Rudner, D.Z. and Losick, R. 2002. A sporulation membrane
protein tethers the pro-K processing enzyme to its inhibitor
and dictates its subcellular localization. Genes & Dev. 16:
1007–1018.
Ryter, A., Schaeffer, P., and Ionesco, H. 1966. [Cytologic classi-
fication, by their blockage stage, of sporulation mutants of
Bacillus subtilis Marburg]. Ann. Inst. Pasteur (Paris) 110:
305–315.
Saleh, O.A., Perals, C., Barre, F.X., and Allemand, J.F. 2004. Fast,
DNA-sequence independent translocation by FtsK in a
single-molecule experiment. EMBO J. 23: 2430–2439.
Selby, C.P. and Sancar, A. 1993. Molecular mechanism of tran-
scription-repair coupling. Science 260: 53–58.
Setlow, P. 1995. Mechanisms for the prevention of damage to
DNA in spores of Bacillus species. Annu. Rev. Microbiol. 49:
29–54.
Sharpe, M.E. and Errington, J. 1995. Postseptational chromo-
some partitioning in bacteria. Proc. Natl. Acad. Sci. 92:
8630–8634.
Sharp, M.D. and Pogliano, K. 1999. An in vivo membrane fusion
assay implicates SpoIIIE in the final stages of engulfment
during Bacillus subtilis sporulation. Proc. Natl. Acad. Sci.
96: 14553–14558.
Stragier, P. and Losick, R. 1996. Molecular genetics of sporula-
tion in Bacillus subtilis. Annu. Rev. Genet. 30: 297–341.
Vellanoweth, R.L. and Rabinowitz, J.C. 1992. The influence of
ribosome-binding-site elements on translational efficiency
in Bacillus subtilis and Escherichia coli in vivo. Mol. Micro-
biol. 6: 1105–1114.
Viollier, P.H., Thanbichler, M., McGrath, P.T., West, L., Mee-
wan, M., McAdams, H.H., and Shapiro, L. 2004. Rapid and
sequential movement of individual chromosomal loci to spe-
cific subcellular locations during bacterial DNA replication.
Proc. Natl. Acad. Sci. 101: 9257–9262.
Wang, X., Possoz, C., and Sherratt, D.J. 2005. Dancing around
the divisome: Asymmetric chromosome segregation in
Escherichia coli. Genes & Dev. 19: 2367–2377.
Webb, C.D., Teleman, A., Gordon, S., Straight, A., Belmont, A.,
Lin, D.C., Grossman, A.D., Wright, A., and Losick, R. 1997.
Bipolar localization of the replication origin regions of chro-
mosomes in vegetative and sporulating cells of B. subtilis.
Cell 88: 667–674.
Wu, L.J. and Errington, J. 1994. Bacillus subtilis spoIIIE protein
required for DNA segregation during asymmetric cell divi-
sion. Science 264: 572–575.
Wu, L.J. and Errington, J. 1997. Septal localization of the SpoIIIE
chromosome partitioning protein in Bacillus subtilis. EMBO
J. 16: 2161–2169.
Wu, L.J. and Errington, J. 1998. Use of asymmetric cell division
and spoIIIE mutants to probe chromosome orientation and
organization in Bacillus subtilis. Mol. Microbiol. 27: 777–
786.
Wuite, G.J., Smith, S.B., Young, M., Keller, D., and Bustamante,
C. 2000. Single-molecule studies of the effect of template
tension on T7 DNA polymerase activity. Nature 404: 103–
106.
Youngman, P.J., Perkins, J.B., and Losick, R. 1983. Genetic
transposition and insertional mutagenesis in Bacillus subti-
lis with Streptococcus faecalis transposon Tn917. Proc.
Natl. Acad. Sci. 80: 2305–2309.
Zhang, L., Higgins, M.L., Piggot, P.J., and Karow, M.L. 1996.
Analysis of the role of prespore gene expression in the com-
partmentalization of mother cell-specific gene expression
during sporulation of Bacillus subtilis. J. Bacteriol. 178:
2813–2817.
Zupancic, M.L., Tran, H., and Hofmeister, A.E. 2001. Chromo-
somal organization governs the timing of cell type-specific
gene expression required for spore formation in Bacillus sub-
tilis. Mol. Microbiol. 39: 1471–1481.
SpoIIIE strips proteins off DNA
GENES & DEVELOPMENT 1795
Cold Spring Harbor Laboratory Presson February 20, 2017 - Published bygenesdev.cshlp.orgDownloaded from