Structure and expression of genes for surface proteins in Paramecium.

Surface proteins from 11 antigenic types of Paramecium tetraurelia vary in molecular weight from 251,000 to 308,000. The size of a series of polyadenylated RNAs obtained from these types were correlated with the sizes of the proteins and judged to be the mRNAs for the proteins. The mRNAs were used to identify genomic DNA clones containing complementary sequences. The gene for antigen A was present in one copy per genome, and the data suggest that extensive introns were absent. When restriction enzyme digests of DNA from cultures of paramecia with active and inactive genes were probed with portions of the cloned genes, no evidence for rearrangements or changes in gene dosage was found.

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RESTRICTION ENZYME ANALYSIS AND CLONING OF HIGH MOLECULAR WEIGHT GENOMIC DNA ISOLATED FROM CHLORELLA SOROKINIANA (CHLOROPHYTA)1

Journal of Phycology ◽

10.1111/j.0022-3646.1990.00361.x ◽

1990 ◽

Vol 26 (2) ◽

pp. 361-367 ◽

Cited By ~ 3

Author(s):

J. Mark Cock ◽

Lori L. Roof ◽

Newell F. Boscomb ◽

Charles W. Gehrke ◽

Kenneth C. Kuo ◽

...

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Restriction Enzyme ◽

Genomic Dna ◽

Chlorella Sorokiniana ◽

Enzyme Analysis ◽

Restriction Enzyme Analysis

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Characterization and comparison of genomic DNA clones containing complementary sequences to mRNA from serotype 51D of Paramecium tetraurelia

MGG Molecular & General Genetics ◽

10.1007/bf00328138 ◽

1987 ◽

Vol 208 (3) ◽

pp. 450-456 ◽

Cited By ~ 7

Author(s):

Helmut J. Schmidt

Keyword(s):

Genomic Dna ◽

Paramecium Tetraurelia ◽

Complementary Sequences

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Susceptibility to insulin-dependent diabetes defined by restriction enzyme polymorphism of HLA-D region genomic DNA

Diabetes ◽

10.2337/diabetes.33.10.958 ◽

1984 ◽

Vol 33 (10) ◽

pp. 958-965 ◽

Cited By ~ 15

Author(s):

D. Owerbach ◽

B. Hagglof ◽

A. Lernmark ◽

G. Holmgren

Keyword(s):

Restriction Enzyme ◽

Genomic Dna ◽

Enzyme Polymorphism ◽

Insulin Dependent Diabetes ◽

D Region ◽

Insulin Dependent

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Interaction of high molecular weight kininogen binding proteins on endothelial cells

Thrombosis and Haemostasis ◽

10.1160/th03-07-0471 ◽

2004 ◽

Vol 91 (01) ◽

pp. 61-70 ◽

Cited By ~ 34

Author(s):

Baby Tholanikunnel ◽

Berhane Ghebrehiwet ◽

Allen Kaplan ◽

Kusumam Joseph

Keyword(s):

Molecular Weight ◽

Endothelial Cell ◽

High Molecular Weight ◽

Gel Filtration ◽

Surface Proteins ◽

Factor Xii ◽

High Molecular Weight Kininogen ◽

Dot Blot ◽

Cell Plasma ◽

Molecular Weight Peak

SummaryCell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endothelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

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Cloning and expression of Xylella fastidiosa antigens in Escherichia coli and Erwinia stewartii

Canadian Journal of Microbiology ◽

10.1139/m89-075 ◽

1989 ◽

Vol 35 (4) ◽

pp. 487-491 ◽

Cited By ~ 3

Author(s):

Paul H. Goodwin

Keyword(s):

Escherichia Coli ◽

Molecular Weight ◽

Genomic Dna ◽

Xylella Fastidiosa ◽

Gene Bank ◽

Cloning And Expression ◽

Western Blots ◽

E Coli ◽

Cosmid Vector ◽

Erwinia Stewartii

Xylella fastidiosa DNA, partially digested with Sau3A, was ligated into the cosmid vector, pUCD615. Approximately 4500 ampicillin-resistant Escherichia coli colonies were obtained. The frequency of complementation of leucine auxotrophy in transfected E. coli indicated that the cosmid gene bank was representative of X. fastidiosa genomic DNA. Colonies were lysed directly onto nitrocellulose membranes using a thermo-inducible λ lysogen and screened for expression of X. fastidiosa antigens. Approximately 16.5% of a random sample of clones were found to express X. fastidiosa antigens as determined by Western blots. These proteins comigrated with proteins of X. fastidiosa and ranged in molecular weight from 10 000 to 160 000. Conjugation of several of the plasmids into Erwinia stewartii resulted in expression of the similar molecular weight cloned proteins with similar levels of expression as in E. coli.Key words: Xylella fastidiosa, Pierce's disease, immunological clone screening, thermo-inducible lysogeny.

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Integrated functional genomic analyses of Klinefelter and Turner syndromes reveal global network effects of altered X chromosome dosage

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1910003117 ◽

2020 ◽

Vol 117 (9) ◽

pp. 4864-4873 ◽

Cited By ~ 6

Author(s):

Xianglong Zhang ◽

David Hong ◽

Shining Ma ◽

Thomas Ward ◽

Marcus Ho ◽

...

Keyword(s):

X Chromosome ◽

Network Effects ◽

Molecular Mechanisms ◽

Klinefelter Syndrome ◽

Gene Dosage ◽

Differentially Expressed ◽

Global Network ◽

Potential Candidate ◽

Gene Promoters ◽

Inactive Genes

In both Turner syndrome (TS) and Klinefelter syndrome (KS) copy number aberrations of the X chromosome lead to various developmental symptoms. We report a comparative analysis of TS vs. KS regarding differences at the genomic network level measured in primary samples by analyzing gene expression, DNA methylation, and chromatin conformation. X-chromosome inactivation (XCI) silences transcription from one X chromosome in female mammals, on which most genes are inactive, and some genes escape from XCI. In TS, almost all differentially expressed escape genes are down-regulated but most differentially expressed inactive genes are up-regulated. In KS, differentially expressed escape genes are up-regulated while the majority of inactive genes appear unchanged. Interestingly, 94 differentially expressed genes (DEGs) overlapped between TS and female and KS and male comparisons; and these almost uniformly display expression changes into opposite directions. DEGs on the X chromosome and the autosomes are coexpressed in both syndromes, indicating that there are molecular ripple effects of the changes in X chromosome dosage. Six potential candidate genes (RPS4X,SEPT6,NKRF,CX0rf57,NAA10, andFLNA) for KS are identified on Xq, as well as candidate central genes on Xp for TS. Only promoters of inactive genes are differentially methylated in both syndromes while escape gene promoters remain unchanged. The intrachromosomal contact map of the X chromosome in TS exhibits the structure of an active X chromosome. The discovery of shared DEGs indicates the existence of common molecular mechanisms for gene regulation in TS and KS that transmit the gene dosage changes to the transcriptome.

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Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform

PeerJ ◽

10.7717/peerj.10340 ◽

2020 ◽

Vol 8 ◽

pp. e10340

Author(s):

Pacharaporn Angthong ◽

Tanaporn Uengwetwanit ◽

Wirulda Pootakham ◽

Kanchana Sittikankaew ◽

Chutima Sonthirod ◽

...

Keyword(s):

Molecular Weight ◽

Genomic Dna ◽

Extraction Methods ◽

Marine Organisms ◽

High Quality ◽

Sequencing Platform ◽

Global Food Security ◽

Long Read ◽

Ctab Method ◽

Global Food

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

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