Isolation and Characterization of Teichoic Acid-Like Substance as an Adhesin ofStaphylococcus aureusto HeLa Cells

ABSTRACT Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class I, no internalization and intracellular growth within HeLa cells; class II, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.

Download Full-text

Isolation and characterization of two novel, closely related ATF cDNA clones from HeLa cells

Nucleic Acids Research ◽

10.1093/nar/18.12.3467 ◽

1990 ◽

Vol 18 (12) ◽

pp. 3467-3473 ◽

Cited By ~ 65

Author(s):

Mireille Gaire ◽

Bruno Chatton ◽

Claude Kedinger

Keyword(s):

Hela Cells ◽

Cdna Clones ◽

Isolation And Characterization

Download Full-text

The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)99234-3 ◽

1991 ◽

Vol 266 (16) ◽

pp. 10358-10367

Author(s):

A. Claude ◽

J. Arenas ◽

J. Hurwitz

Keyword(s):

Hela Cells ◽

Rna Helicase ◽

Isolation And Characterization ◽

Nuclear Extracts

Download Full-text

The structure of the extracellular teichoic acids from the allergy-protective bacterium Lactococcus lactis G121

Biological Chemistry ◽

10.1515/hsz-2012-0142 ◽

2012 ◽

Vol 393 (8) ◽

pp. 749-755

Author(s):

Kathleen Fischer ◽

Evgueny Vinogradov ◽

Buko Lindner ◽

Holger Heine ◽

Otto Holst

Keyword(s):

Lactococcus Lactis ◽

Molecular Mechanisms ◽

Cell Envelope ◽

Teichoic Acid ◽

Size Exclusion ◽

Resonance Spectroscopy ◽

Protective Properties ◽

Isolation And Characterization ◽

Exclusion Chromatography

Abstract The Gram-positive bacterium Lactococcus lactis G121 is a farm isolate that protects mice from ovalbumin-induced asthma. To understand the molecular mechanisms of such allergy-protective properties, the isolation and characterization of cell envelope constituents is crucial. Here, structural analyses of the extracellular teichoic acid (EC TA) from L. lactis G121 are presented. Extraction with 0.9% saline afforded a crude TA fraction. Consecutive size exclusion chromatography on Biogel P60 and P10 matrix was performed to purify the sample. Chemical component analyses, high-resolution electrospray ionization Fourier-transformed ion cyclotron mass spectrometry, and nuclear magnetic resonance spectroscopy were conducted for structural elucidation. The EC TA was a poly(glycosylglycerol phosphate) molecule with a repeating unit of -6)-[β-d-Glcp-(1→3)-][α-d-GlcpNAc-(1→4)-]α-d-GalpNAc-(1→3)-β-d-GlcpNAc-(1→2)-glycerol-(1-P-).

Download Full-text

Isolation and characterization of an RNA ligase from HeLa cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.82.3.684 ◽

1985 ◽

Vol 82 (3) ◽

pp. 684-688 ◽

Cited By ~ 29

Author(s):

K. K. Perkins ◽

H. Furneaux ◽

J. Hurwitz

Keyword(s):

Hela Cells ◽

Rna Ligase ◽

Isolation And Characterization

Download Full-text

Isolation and Characterization of Connexin26 Gap Junctions from Transfected Hela Cells

Microscopy and Microanalysis ◽

10.1017/s1431927600033729 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 246-247

Author(s):

G. Hand ◽

V. Nguyen ◽

K. Downing ◽

D. Mueller ◽

A. Engel ◽

...

Keyword(s):

Amino Acid ◽

Gap Junctions ◽

Hela Cells ◽

Multigene Family ◽

Similar Sequence ◽

Isolation And Characterization ◽

Extracellular Loops ◽

Junction Structure ◽

Better Than

Gap junctions are composed of a multigene family of proteins called the connexins with a similar sequence and folding topology. Connexons, half-channels made from a particular connexin, will dock and form functional channels with some, but not all, connexons made of other isoforms. This is surprising considering that the primary sequences of the docking domains are conserved and in some cases there are limited amino acid changes in the extracellular loops between compatible connexins. Therefore, some tertiary or quaternary interactions between the extracellular loops of the two docking hemichannels must contribute to the compatibility of a connexon assembled from one connexin for a connexon made from a different connexin. The 3D structures of gap junctions composed of only three isoforms have been investigated with only one case of a gap junction structure at better than 1 nm resolution [1]. The rat liver hemichannel structure is the only one that contains an exposed extracellular surface [2].

Download Full-text

The mouse androgen receptor. Functional analysis of the protein and characterization of the gene

Biochemical Journal ◽

10.1042/bj2780269 ◽

1991 ◽

Vol 278 (1) ◽

pp. 269-278 ◽

Cited By ~ 28

Author(s):

P W Faber ◽

A King ◽

H C J van Rooij ◽

A O Brinkmann ◽

N J de Both ◽

...

Keyword(s):

Androgen Receptor ◽

Reporter Gene ◽

Hela Cells ◽

Transcription Initiation ◽

Promoter Region ◽

Sequence Similarity ◽

Dependent Manner ◽

S1 Nuclease ◽

Isolation And Characterization

Screening a mouse genomic DNA library with human androgen-receptor (hAR) cDNA probes resulted in the isolation and characterization of eight genomic fragments that contain the eight exons of the mouse androgen-receptor (mAR) gene. On the basis of similarity to the hAR gene, the nucleotide sequences of the protein-coding parts of the exons as well as the sequences of the intron/exon boundaries were determined. An open reading frame (ORF) of 2697 nucleotides, which can encode an 899-amino-acid protein, could be predicted. The structure of the mAR ORF was confirmed by sequence analysis of mAR cDNA fragments, which were obtained by PCR amplification of mouse testis cDNA, using mAR specific primers. A eukaryotic mAR expression vector was constructed and mAR was transiently expressed in COS-1 cells. The expressed protein was shown by Western blotting to be identical in size with the native mAR. Co-transfection of HeLa cells with the mAR expression plasmid and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter-gene construct showed mAR to be able to trans-activate the androgen-responsive promoter in a ligand-dependent manner. Transcription-initiation sites of the mAR gene were identified by S1-nuclease protection experiments, and the functional activity of the promoter region was determined by transient expression of mAR promoter-CAT-reporter-gene constructs in HeLa cells. Structural analysis revealed the promoter of the mAR gene to be devoid of TATA/CCAAT elements. In addition, the promoter region is not remarkably (G + C)-rich. Potential promoter elements consist of a consensus Sp1 binding sequence and a homopurine stretch. The polyadenylation sites of mAR mRNA were identified by sequence similarity to the corresponding sites in the hAR mRNA.

Download Full-text