scholarly journals Separation of Micronuclear DNA of Stylonychia lemnae by Pulsed-Field Electrophoresis and Identification of a DNA Molecule with a High Copy Number

1999 ◽  
Vol 9 (7) ◽  
pp. 654-661
Author(s):  
Christian Maercker ◽  
Heike Kortwig ◽  
Hans J. Lipps
Keyword(s):  
Copy Number ◽  
High Copy Number ◽  
Pulsed Field ◽  
Macronuclear Development ◽  
Dna Molecule ◽  
Pulsed Field Electrophoresis ◽  
Dna Fragment ◽  
Nuclear Differentiation ◽  
Excellent Tool ◽  
Very High

DNA from the hypotrichous ciliatae Stylonychia lemnae was separated by PFGE. In addition to the separation of the macronuclear DNA molecules with a size up to ∼40 kb, we were able to separate the micronuclear DNA with a size between ∼90 kb and 2 Mb. One very prominent 90-kb DNA band appeared on the pulsed-field gels. We propose that this 90-kb DNA fragment represents a linear plasmid residing in the micronucleus in a very high copy number. About 10% of the micronuclear DNA consists of the 90-kb DNA molecule. It appears in the micronucleus as well as in the macronuclear anlagen during macronuclear development but not in the mature macronucleus. Thus, the multicopy DNA is eliminated during fragmentation of the macronuclear anlagen DNA in the course of macronuclear development. Therefore, this 90-kb DNA molecule might serve as an excellent tool to study the recognition and elimination of DNA during nuclear differentiation of hypotrichous ciliates.

Isolation of a DNA fragment that is expressed as an amber suppressor when present in high copy number in yeast

Gene ◽  
1984 ◽  
Vol 29 (1-2) ◽  
pp. 69-76 ◽  
Author(s):  
Isabel L. Calderon ◽  
C.Rebecca Contopoulou ◽  
Robert K. Mortimer
Keyword(s):  
Copy Number ◽  
High Copy Number ◽  
Amber Suppressor ◽  
Dna Fragment

scholarly journals HiCancer: accurate and complete cancer genome phasing with Hi-C reads

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Weihua Pan ◽  
Desheng Gong ◽  
Da Sun ◽  
Haohui Luo
Keyword(s):  
Copy Number ◽  
Somatic Mutations ◽  
Copy Number Variations ◽  
Cancer Genome ◽  
Structural Variations ◽  
Genome Map ◽  
Linkage Information ◽  
Suboptimal Solution ◽  
Allelic Copy Number ◽  
Very High

AbstractDue to the high complexity of cancer genome, it is too difficult to generate complete cancer genome map which contains the sequence of every DNA molecule until now. Nevertheless, phasing each chromosome in cancer genome into two haplotypes according to germline mutations provides a suboptimal solution to understand cancer genome. However, phasing cancer genome is also a challenging problem, due to the limit in experimental and computational technologies. Hi-C data is widely used in phasing in recent years due to its long-range linkage information and provides an opportunity for solving the problem of phasing cancer genome. The existing Hi-C based phasing methods can not be applied to cancer genome directly, because the somatic mutations in cancer genome such as somatic SNPs, copy number variations and structural variations greatly reduce the correctness and completeness. Here, we propose a new Hi-C based pipeline for phasing cancer genome called HiCancer. HiCancer solves different kinds of somatic mutations and variations, and take advantage of allelic copy number imbalance and linkage disequilibrium to improve the correctness and completeness of phasing. According to our experiments in K562 and KBM-7 cell lines, HiCancer is able to generate very high-quality chromosome-level haplotypes for cancer genome with only Hi-C data.

Genetic interactions between CDC31 and KAR1, two genes required for duplication of the microtubule organizing center in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 407-422 ◽  
Author(s):  
E A Vallen ◽  
W Ho ◽  
M Winey ◽  
M D Rose
Keyword(s):  
Copy Number ◽  
Gene Dosage ◽  
Spindle Pole Body ◽  
Direct Interaction ◽  
Spindle Pole ◽  
High Copy Number ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Recessive Mutations ◽  
Organizing Center

Abstract KAR1 encodes an essential component of the yeast spindle pole body (SPB) that is required for karyogamy and SPB duplication. A temperature-sensitive mutation, kar1-delta 17, mapped to a region required for SPB duplication and for localization to the SPB. To identify interacting SPB proteins, we isolated 13 dominant mutations and 3 high copy number plasmids that suppressed the temperature sensitivity of kar1-delta 17. Eleven extragenic suppressor mutations mapped to two linkage groups, DSK1 and DSK2. The extragenic suppressors were specific for SPB duplication and did not suppress karyogamy-defective alleles. The major class, DSK1, consisted of mutations in CDC31. CDC31 is required for SPB duplication and encodes a calmodulin-like protein that is most closely related to caltractin/centrin, a protein associated with the Chlamydomonas basal body. The high copy number suppressor plasmids contained the wild-type CDC31 gene. One CDC31 suppressor allele conferred a temperature-sensitive defect in SPB duplication, which was counter-suppressed by recessive mutations in KAR1. In spite of the evidence for a direct interaction, the strongest CDC31 alleles, as well as both DSK2 alleles, suppressed a complete deletion of KAR1. However, the CDC31 alleles also made the cell supersensitive to KAR1 gene dosage, arguing against a simple bypass mechanism of suppression. We propose a model in which Kar1p helps localize Cdc31p to the SPB and that Cdc31p then initiates SPB duplication via interaction with a downstream effector.

Characterization of High-Copy-Number Retrotransposons From the Large Genomes of the Louisiana Iris Species and Their Use as Molecular Markers

Genetics ◽  
2003 ◽  
Vol 164 (2) ◽  
pp. 685-697 ◽  
Author(s):  
Edward K Kentner ◽  
Michael L Arnold ◽  
Susan R Wessler
Keyword(s):  
Molecular Markers ◽  
Copy Number ◽  
High Copy Number ◽  
Marker System ◽  
Transposon Display ◽  
Plant Genomes ◽  
Large Plant ◽  
Backcross Hybrids ◽  
Abundance And Distribution

Abstract The Louisiana iris species Iris brevicaulis and I. fulva are morphologically and karyotypically distinct yet frequently hybridize in nature. A group of high-copy-number TY3/gypsy-like retrotransposons was characterized from these species and used to develop molecular markers that take advantage of the abundance and distribution of these elements in the large iris genome. The copy number of these IRRE elements (for iris retroelement), is ∼1 × 105, accounting for ∼6–10% of the ∼10,000-Mb haploid Louisiana iris genome. IRRE elements are transcriptionally active in I. brevicaulis and I. fulva and their F1 and backcross hybrids. The LTRs of the elements are more variable than the coding domains and can be used to define several distinct IRRE subfamilies. Transposon display or S-SAP markers specific to two of these subfamilies have been developed and are highly polymorphic among wild-collected individuals of each species. As IRRE elements are present in each of 11 iris species tested, the marker system has the potential to provide valuable comparative data on the dynamics of retrotransposition in large plant genomes.

Plasmid pMTL153: a high copy number version of pAT153 and its use to obtain high expression of the Pseudomonas carboxypeptidase G2 gene

1988 ◽  
Vol 29 (6) ◽  
pp. 572-578 ◽  
Author(s):  
Steve P. Chambers ◽  
Sue E. Prior ◽  
Rachell A. Evans ◽  
Roger F. Sherwood ◽  
Nigel P. Minton
Keyword(s):  
Copy Number ◽  
High Expression ◽  
High Copy Number ◽  
Carboxypeptidase G2

Influence of the agarose matrix in pulsed-field electrophoresis

1993 ◽  
Vol 14 (1) ◽  
pp. 349-354 ◽  
Author(s):  
Francis H. Kirkpatrick ◽  
Maxine M. Dumais ◽  
Hugh W. White ◽  
Kenneth B. Guiseley
Keyword(s):  
Pulsed Field ◽  
Pulsed Field Electrophoresis

scholarly journals Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

2015 ◽  
Vol 82 (4) ◽  
pp. 1286-1294 ◽  
Author(s):  
Evelyn Durmaz ◽  
Yan Hu ◽  
Raffi V. Aroian ◽  
Todd R. Klaenhammer
Keyword(s):  
Bacillus Thuringiensis ◽  
Lactococcus Lactis ◽  
Copy Number ◽  
Oral Delivery ◽  
Membrane Integrity ◽  
Biologically Active ◽  
High Copy Number ◽  
Cry Protein ◽  
Western Blots ◽  
Content Type

ABSTRACTTheBacillus thuringiensiscrystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) inLactococcus lactisfor potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production,cry5Bwas cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes inLactococcuslysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strainL. lactisKP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates fromL. lactiscultures expressing both Cry5B and tCry5B,in vivochallenges ofCaenorhabditis elegansworms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly fromL. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.

Isolation of STD1, a high-copy-number suppressor of a dominant negative mutation in the yeast TATA-binding protein

1993 ◽  
Vol 13 (6) ◽  
pp. 3650-3659
Author(s):  
R W Ganster ◽  
W Shen ◽  
M C Schmidt
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Copy Number ◽  
Slow Growth ◽  
Tata Binding Protein ◽  
Dominant Negative ◽  
High Copy Number ◽  
N Terminus ◽  
Growth Phenotype

The TATA-binding protein (TBP) is an essential component of the transcriptional machinery of all three nuclear RNA polymerase enzymes. Comparison of the amino acid sequence of TBPs from a number of species reveals a highly conserved 180-residue C-terminal domain. In contrast, the N terminus is variable in both size and amino acid sequence. Overexpression of a TBP protein with a deletion of the nonconserved N terminus (TBP delta 57) in Saccharomyces cerevisiae results in a dominant negative phenotype of extremely slow growth. Associated with the slow-growth phenotype are defects in RNA polymerase II transcription in vivo. We have screened a high-copy-number yeast genomic library for suppression of the slow-growth phenotype and have isolated plasmids which encode suppressors of TBP delta 57 overexpression. Here we report the sequence and initial characterization of one suppressor, designated STD1 for suppressor of TBP deletion. The STD1 gene contains a single continuous open reading frame with the potential to encode a 50.2-kDa protein. Disruption of the STD1 gene indicates that it is not essential for vegetative growth, mating, or sporulation. High-copy-number suppression by the STD1 gene is not the result of a decrease in TBP delta 57 protein accumulation or DNA-binding activity; instead, STD1 suppression is coincident with the elimination of TBP delta 57-induced RNA polymerase II defects in both uninduced and induced transcription in vivo.

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