scholarly journals Flanking Regulatory Sequences of theTetrahymena R Deletion Element Determine the Boundaries of DNA Rearrangement

1999 ◽  
Vol 19 (8) ◽  
pp. 5631-5641 ◽  
Author(s):  
Douglas L. Chalker ◽  
Antonietta La Terza ◽  
Allison Wilson ◽  
Christopher D. Kroenke ◽  
Meng-Chao Yao
Keyword(s):  
Sequence Similarity ◽  
Common Mechanism ◽  
Complex Nature ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Flanking Region ◽  
The Many ◽  
The Right ◽  
Flanking Regions

ABSTRACT In the ciliate Tetrahymena thermophila, thousands of DNA segments of variable size are eliminated from the developing somatic macronucleus by specific DNA rearrangements. It is unclear whether rearrangement of the many different DNA elements occurs via a single mechanism or via multiple rearrangement systems. In this study, we characterized in vivo cis-acting sequences required for the rearrangement of the 1.1-kbp R deletion element. We found that rearrangement requires specific sequences flanking each side of the deletion element. The required sequences on the left side appear to span roughly a 70-bp region that is located at least 30 bp from the rearrangement boundary. When we moved the location of the leftcis-acting sequences closer to the eliminated region, we observed a rightward shift of the rearrangement boundary such that the newly formed deletion junction retained its original distance from this flanking region. Likewise, when we moved the flanking region as much as 500 bp away from the deletion element, the rearrangement boundary shifted to remain in relative juxtaposition. Clusters of base substitutions made throughout this critical flanking region did not affect rearrangement efficiency or accuracy, which suggests a complex nature for this regulatory sequence. We also found that the right flanking region effectively replaced the essential sequences identified on the left side, and thus, the two flanking regions contain sequences of analogous function despite the lack of obvious sequence identity. These data taken together indicate that the R-element flanking regions contain sequences that position the rearrangement boundaries from a short distance away. Previously, a 10-bp polypurine tract flanking the M-deletion element was demonstrated to act from a distance to determine its rearrangement boundaries. No apparent sequence similarity exists between the M and R elements. The functional similarity between these different cis-acting sequences of the two elements is firm support for a common mechanism controlling Tetrahymenarearrangement.

scholarly journals Evolution of lineage-specific functions in ancient cis -regulatory modules

Open Biology ◽  
2015 ◽  
Vol 5 (11) ◽  
pp. 150079 ◽  
Author(s):  
Stefan Pauls ◽  
Debbie K. Goode ◽  
Libero Petrone ◽  
Paola Oliveri ◽  
Greg Elgar
Keyword(s):  
Gene Expression ◽  
Morphological Evolution ◽  
Regulatory Sequences ◽  
Enhancer Activity ◽  
Regulatory Modules ◽  
History Of ◽  
Flanking Region ◽  
Regulatory Functions ◽  
Conserved Core ◽  
Flanking Regions

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis -regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.

DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

scholarly journals DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898 ◽  
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

scholarly journals Analysis of Human Cytomegalovirus oriLyt Sequence Requirements in the Context of the Viral Genome

2005 ◽  
Vol 79 (6) ◽  
pp. 3615-3626 ◽  
Author(s):  
Eva-Maria Borst ◽  
Martin Messerle
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Core Region ◽  
Origin Of Replication ◽  
Flanking Region ◽  
The Right ◽  
Flanking Regions ◽  
Sequence Requirements ◽  
Replication Activity

ABSTRACT During the lytic phase of infection, replication of herpesvirus genomes initiates at the lytic origin of replication, oriLyt. Many herpesviruses harbor more than one lytic origin, but so far, only one oriLyt has been identified for human cytomegalovirus (HCMV). Evidence for the existence of additional lytic origins of HCMV has remained elusive. On the basis of transient replication assays with cloned viral fragments, HCMV oriLyt was described as a core region of 1.5 kbp (minimal oriLyt) flanked by auxiliary sequences required for maximal replication activity (complete oriLyt). It remained unclear whether minimal oriLyt alone can drive the replication of HCMV in the absence of its accessory regions. To investigate the sequence requirements of oriLyt in the context of the viral genome, mutant genomes were constructed lacking either minimal or complete oriLyt. These genomes were not infectious, suggesting that HCMV contains only one lytic origin of replication. Either minimal or complete oriLyt was then ectopically reinserted into the oriLyt-depleted genomes. Only the mutant genomes carrying complete oriLyt led to infectious progeny. Remarkably, inversion of the 1.5-kbp core origin relative to its flanking regions resulted in a replication-defective genome. Mutant genomes carrying minimal oriLyt plus the left flanking region gave rise to minifoci, but genomes harboring minimal oriLyt together with the right flanking region were noninfectious. We conclude that the previously defined minimal lytic origin is not sufficient to drive replication of the HCMV genome. Rather, our results underline the importance of the accessory regions and their correct arrangement for the function of HCMV oriLyt.

scholarly journals Structural and Functional Analysis of Rv3214 from Mycobacterium tuberculosis, a Protein with Conflicting Functional Annotations, Leads to Its Characterization as a Phosphatase

2006 ◽  
Vol 188 (10) ◽  
pp. 3589-3599 ◽  
Author(s):  
Harriet A. Watkins ◽  
Edward N. Baker
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Functional Annotation ◽  
Sequence Similarity ◽  
Phosphoglycerate Mutase ◽  
Phosphopantetheinyl Transferase ◽  
Ph Optimum ◽  
Reading Frame ◽  
Functional Annotations ◽  
The Many

ABSTRACT The availability of complete genome sequences has highlighted the problems of functional annotation of the many gene products that have only limited sequence similarity with proteins of known function. The predicted protein encoded by open reading frame Rv3214 from the Mycobacterium tuberculosis H37Rv genome was originally annotated as EntD through sequence similarity with the Escherichia coli EntD, a 4′-phosphopantetheinyl transferase implicated in siderophore biosynthesis. An alternative annotation, based on slightly higher sequence identity, grouped Rv3214 with proteins of the cofactor-dependent phosphoglycerate mutase (dPGM) family. The crystal structure of this protein has been solved by single-wavelength anomalous dispersion methods and refined at 2.07-Å resolution (R = 0.229; Rfree = 0.245). The protein is dimeric, with a monomer fold corresponding to the classical dPGM α/β structure, albeit with some variations. Closer comparisons of structure and sequence indicate that it most closely corresponds with a broad-spectrum phosphatase subfamily within the dPGM superfamily. This functional annotation has been confirmed by biochemical assays which show negligible mutase activity but acid phosphatase activity with a pH optimum of 5.4 and suggests that Rv3214 may be important for mycobacterial phosphate metabolism in vivo. Despite its weak sequence similarity with the 4′-phosphopantetheinyl transferases (EntD homologues), there is little evidence to support this function.

scholarly journals Human Asset Accounting And Measurement: Moving Forward

2014 ◽  
Vol 12 (2) ◽  
pp. 93
Author(s):  
Brian B. Stanko ◽  
Thomas L. Zeller ◽  
Matthew F. Melena
Keyword(s):  
Human Capital ◽  
Financial Reporting ◽  
Sustainability Reporting ◽  
Accurate Method ◽  
Complex Nature ◽  
Substantial Part ◽  
Universal Method ◽  
History Of ◽  
The Many ◽  
The Right

Changes in the accounting profession and in the way organizations are managed and operated over the past several decades have led to the identification of a new factor that makes up a substantial part of the value of an organization: human capital. The value that employees add to organizations, however, has been difficult to measure because of the many elements that comprise it and aspects of human nature and free will that are involved. Many models have been proposed to capture the value that organizations gain from employees, but none have succeeded in full. Additionally, strict financial reporting regulations would require an accurate and uniform method of accounting for human capital in order to give much relevance to the data collected. Despite its complex nature, the field of human asset accounting continues to gain momentum and is headed in the right direction. The development of a universal method of accounting for human capital would provide a much more exact valuation of organizations and have deep benefits for owners, managers, investors, accountants, and human resource employees. This paper examines the history of human asset accounting and its feasibility in current financial reporting environments. Additionally, it demonstrates the importance of human asset accounting, different approaches toward human asset accounting, and how beneficial an accurate method could prove to be in financial reporting. Finally, the paper recommends that, as a precursor to measurement, the development of general quantitative and qualitative human capital disclosures, with real company examples, be included in a companys sustainability reporting.

Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element

1991 ◽  
Vol 11 (3) ◽  
pp. 1334-1343
Author(s):  
R Paciucci ◽  
A Pellicer
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Transcription Unit ◽  
Regulatory Sequences ◽  
Hypersensitive Site ◽  
Ras Gene ◽  
Rnase Protection ◽  
Negative Regulatory Element ◽  
Flanking Region

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.

CHARACTERIZATION OFTHE 5’FLANKING REGION FOR THE HUMAN FIBRINOGEN β GENE

1987 ◽  
Author(s):  
P Huber ◽  
J Dalmon ◽  
M Laurent ◽  
G Courtois ◽  
D Thevenon ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Genomic Library ◽  
Hepatoma Cell ◽  
Regulatory Sequence ◽  
Hepatoma Cell Line ◽  
Regulatory Sequences ◽  
Potential Candidate ◽  
Human Fibrinogen ◽  
Flanking Region

Fibrinogen is coded by three separate genes located in a 50kb region of chromosome 4 and organized in a α - β - γ orientation with an inversion of the gene 3- A human genomic library was constructed using the EMBL4 phage and screened with cDNA probes coding for human fibrinogen Aα, Bβ and γ chains. Clones, covering the fibrinogen locus,were identified, and their organization was analyzed by means of hybridization and restriction mapping. Among these clones one recombinant phage containing the β gene and large 5’ and 3’ -flanking sequences was isolated.To identify the regulatory sequences Dpstream from the human β gene, a 1.5 kb fragment of the immediate 5’-flanking region was sequenced. The SI mapping experiments revealed three transcription initiation sites. PotentialTATA and CAAT sequences were identified upstream the initiation start points at the positions -21 and -58 from the first initiation start point.Comparison of this sequence with that previously reported for the same region upstream from the human γ gene revealed no significant homology which suggests that the potential promoting sequences of these genes are different. In contrast, comparison of the 5’flanking regions of human and rat β genes showed more than 80% homology for 142 bp upstream from the gene. This highly conserved region is a potential candidate for a regulatory sequence of the human β gene.To verify this activity, a β fibrinogen minigene was constructed by deletion of the internal part of the normal gene and including 3.4kb of the 5’flanking region and 1.4kb of the 3’flanking region. The minigene was transfected into HepG2, a human hepatoma cell line, to show whether the 5’flanking region of the human fibrinogen gene contains DNA sequences sufficient for efficient transcription in HepG2. Constructions of several parts of the sequenced 5’flanking region of the human β gene with the gene of the chloramphenical acetyl transferase have been also transfected in the HepG2 cells to determine the specificity of the gene expression and to localize the sequences controlling the transcription of the gene.

scholarly journals Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element.

1991 ◽  
Vol 11 (3) ◽  
pp. 1334-1343 ◽  
Author(s):  
R Paciucci ◽  
A Pellicer
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Transcription Unit ◽  
Regulatory Sequences ◽  
Hypersensitive Site ◽  
Ras Gene ◽  
Rnase Protection ◽  
Negative Regulatory Element ◽  
Flanking Region

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.

scholarly journals Structural and Functional Differences in the Promoter and 5′ Flanking Region of Ldh-B Within and Between Populations of the Teleost Fundulus heteroclitus

Genetics ◽  
1997 ◽  
Vol 145 (3) ◽  
pp. 759-769
Author(s):  
Patricia M Schulte ◽  
Marta Gómez-Chiarri ◽  
Dennis A Powers
Keyword(s):  
Reporter Gene ◽  
Fundulus Heteroclitus ◽  
Cultured Cells ◽  
Functional Differentiation ◽  
Flanking Sequence ◽  
Tests Of Neutrality ◽  
Functional Consequences ◽  
Flanking Region ◽  
Flanking Regions

We have investigated the mechanisms underlying differences in the transcriptional regulation of lactate dehydrogenase-B (Ldh-B) between northern and southern populations of a teleost fish, Fundulus heteroclitus. A 1-kb region immediately 5′ of the gene was sequenced from populations throughout the species range. There were two major allele classes in the sample, one containing alleles from Maine and another containing those from Florida. Populations from intermediate localities contained both allele classes. Some individuals from Georgia had sequences intermediate between the two classes, representing either ancestral alleles or recombinants. Tests of neutrality were applied to determine whether observed variation was consistent with neutral expectations. Significant deviations from neutral expectations were detected for the 5′ flanking region, but not for other loci. The functional consequences of flanking sequence variation were assessed by transfection of reporter gene constructs into cultured cells and injection into living fish. Consistent with observed variation in Ldh-B transcription rate between populations, significant differences in reporter gene activity were driven by flanking regions from northern and southern populations both in cell culture and in vivo. This functional differentiation, coupled with departures from neutral expectations, suggests that selection may have acted on the regulation of Ldh-B in F. heteroclitus.

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