CHARACTERIZATION OF THE GENE FOR HUMAN HISTIDINE-RICH GLYCOPROTEIN

1987 ◽  
Author(s):  
T Koide
Keyword(s):  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Single Chain ◽  
Structural Domains ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Human Genomic ◽  
Considerable Homology ◽  
Amino Acid Segment

Human histidine-rich glycoprotein (HRG) is a single-chain glycoprotein in plasma which is considered to modulate a coagulation and fibrinolysis system with the ability to bind to heparin, plasminogen, fibrinogen, thrombospondin, etc. Recently we have elucidated the primary structure of HRG by determining the nucleotide sequence of its cDNA, and showed that HRG is composed of several different types of internal repeats, each one of which shows considerable homology with the functional and/or structural domains of other proteins including high molecular weight kininogen, antithrombin III, cystatins, and proline-rich protein and peptide. Thus, the multifunctional property of HRG was suggested to be due to its multi-domain structure. In the present studies, a human genomic DNA library, cloned in the bacteriophage vector Charon 4A, was screened for HRG gene using a full-length cDNA coding for human IMI as a probe. A total of 7 clones were isolated from 6 × 105 phage and each was plaque purified. The entire HRG gene is represented in 3 genomic inserts with overlapping sequences that carry human DNA spanning 30 kb. Overlapping gene fragments were subcloned into pUC9 and characterized by Southern blot hybridization using 5’ and 3’ end probes isolated from human HRG cDNA and by DNA sequencing. These studies have shown that the gene for human HRG spans about 9 kb and consists of at least 5 exons and 4 introns. The putative histidine-rich region consisted of 12 tandemly repeated sequences of a 5 amino acid segment and 2 proline-rich regions contiguous to it are likely to be involved within one exon.

Eimeria species: studies using rRNA and rDNA probes

Parasitology ◽  
1990 ◽  
Vol 101 (1) ◽  
pp. 1-6 ◽  
Author(s):  
J. Ellis ◽  
J. Bumstead
Keyword(s):  
Genomic Dna ◽  
Southern Hybridization ◽  
Eimeria Species ◽  
Small Subunit ◽  
Rrna Genes ◽  
Rdna Probe ◽  
Dna Library ◽  
Rdna Sequences ◽  
Genomic Dna Library

SUMMARYrRNA and a heterologous cloned rDNA probe have been used to detect the rRNA genes of Eimeria species which infe the chicken, and has allowed the isolation and preliminary characterization of cloned rDNA sequences from a genomic DNA library of Eimeria tenella. It is demonstrated that rRNA and rDNA probes can be used to identify individual Eimeria species by the restriction fragment patterns detected after Southern hybridization. In addition, studies have shown that the large and small subunit rRNAs are expressed throughout sporulation.

scholarly journals Characterization of two BHV-4 strains isolated in the Czech Republic

2010 ◽  
Vol 55 (No. 3) ◽  
pp. 106-112 ◽  
Author(s):  
V. Fichtelova ◽  
K. Kovarcik
Keyword(s):  
Reference Strain ◽  
Fragment Size ◽  
Blot Hybridization ◽  
Size Variation ◽  
Southern Blot Hybridization ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
The Czech Republic ◽  
Herpesvirus 4

This study describes the isolation of bovine herpesvirus 4 (BHV-4) from the respiratory tract of animals suffering from respiratory disease. DNA of new isolates, CH and Ni, was cleaved with <I>Bam</I>HI, <I>Eco</I>RI and <I>Hind</I>III in restriction enzyme analysis and the fragments were identified by co-migration with the restriction profile of the reference strain Movar 33/63 cleaved with the appropriate endonuclease. Typical profiles with polyrepetitive DNA (prDNA) fragments were detected. In order to localize the size variation within the obtained digestion fragments, Southern blot hybridization was performed. Differences between the isolates CH, Ni were localized in both the prDNAs and the unique central part of the genome and were restricted to fragment size variation.

Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture

2020 ◽  
Author(s):  
Kyriaki Xanthopoulou ◽  
Julia Wille ◽  
Janine Zweigner ◽  
Kai Lucaßen ◽  
Thorsten Wille ◽  
...  
Keyword(s):  
Blood Culture ◽  
Enterococcus Faecium ◽  
Core Genome ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Vana Gene ◽  
Plasmid Analysis ◽  
Vancomycin Resistant ◽  
Faecium Isolate

Abstract Objectives To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. Methods The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Results Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Conclusions Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

scholarly journals Freshwater Bacteria Can Methylate Selenium through the Thiopurine Methyltransferase Pathway

2003 ◽  
Vol 69 (7) ◽  
pp. 3784-3790 ◽  
Author(s):  
Lionel Ranjard ◽  
Sylvie Nazaret ◽  
Benoit Cournoyer
Keyword(s):  
Pseudomonas Syringae ◽  
Sodium Selenite ◽  
Blot Hybridization ◽  
Sodium Selenate ◽  
Thiopurine Methyltransferase ◽  
Freshwater Environment ◽  
Reading Frame ◽  
Dna Library ◽  
Freshwater Bacteria ◽  
Genomic Dna Library

ABSTRACT Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated. A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used. The distribution of the bTPMT gene (tpm) in the microflora was studied. Freshwater bacteria growing on 10 μM sodium selenite and 10 μM sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe. Ribotyping showed that these strains are closely related. The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed. Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence. The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe. The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing. BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P. syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71%. PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed. E. coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation. The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated. An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine. These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes.

scholarly journals Characterization of tetracycline and erythromycin resistance determinants in Treponema denticola.

1996 ◽  
Vol 40 (7) ◽  
pp. 1690-1694 ◽  
Author(s):  
M C Roberts ◽  
W O Chung ◽  
D E Roe
Keyword(s):  
Host Range ◽  
Enterococcus Faecalis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Culture Collection ◽  
Treponema Denticola ◽  
Erythromycin Resistance ◽  
Resistance Determinants ◽  
Pcr Assays

Treponema denticola isolates were evaluated for the presence of known tetracycline and erythromycin resistance determinants by Southern blot hybridization of whole-cell DNA and PCR assays. We examined all isolates available, which included 12 clinical and 4 American Type Culture Collection isolates. Two isolates carried the Tet B determinant, five isolates carried both the Tet B and Erm F determinants, seven isolates carried the Erm F determinant, and two did not carry any of the Tet or Erm determinants tested. Both the Tet B and Erm F determinants appeared to be associated with the chromosome. Neither of the two T. denticola donors tested could transfer the Tet B determinant, but three of four T. denticola tested transferred the Erm F determinant to an Enterococcus faecalis recipient. This extends the host range of both the tetB and ermF genes into the genus Treponema.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome

1986 ◽  
Vol 6 (12) ◽  
pp. 4516-4525
Author(s):  
K G Coleman ◽  
H Y Steensma ◽  
D B Kaback ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
Genomic Dna ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Yeast Genome ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library ◽  
Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

Microisolation of the chicken Z chromosome and construction off microclone libraries

Genome ◽  
1997 ◽  
Vol 40 (6) ◽  
pp. 865-872 ◽  
Author(s):  
Régis Zimmer ◽  
Alon Haberfeld ◽  
Ann M. Verrinder Gibbins
Keyword(s):  
Light Microscope ◽  
Genomic Dna ◽  
Size Range ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Z Chromosome ◽  
Mitotic Metaphase ◽  
Simple Method ◽  
Dna Library ◽  
Male Chicken

A simple method was used to adapt a standard light microscope for the collection of chicken Z chromosomes from mitotic-metaphase spreads. The DNA of the collected chromosomes was enzymatically amplified using a partially degenerate primer. The resulting sequences, within a size range of 200–800 bp, were cloned to produce a Z chromosome DNA library, using blunt-end ligation into a SmaI-digested pUC18 plasmid (the SureClone system; Pharmacia, U.S.A.). The microcloning experiments produced 1250 clones; the size range of the cloned inserts was 250–800 bp, with an average of 480 bp (176 clones examined). Using male chicken genomic DNA as a probe, 10 out of 17 randomly selected clones showed strong positive signals on Southern blots, confirming the origin of the inserts as chicken DNA. In addition, the Z-chromosome origin of a selected microclone was verified in a semiquantitative Southern blot hybridization that showed positive signals with intensities that were approximately twice as strong for male (ZZ) as for female (ZW) chicken genomic DNA when the clone was used as a probe. The value of these libraries in further analysis of the chicken Z chromosome is discussed.Key words: microdissection, microcloning, chicken Z chromosome.

Characterization of a mouse multigene family that encodes zinc finger structures

1988 ◽  
Vol 8 (3) ◽  
pp. 1319-1326
Author(s):  
P Chavrier ◽  
P Lemaire ◽  
O Revelant ◽  
R Bravo ◽  
P Charnay
Keyword(s):  
Zinc Finger ◽  
Large Family ◽  
Nucleotide Sequence Analysis ◽  
Transcriptional Level ◽  
Cross Hybridization ◽  
Dna Library ◽  
Genes Encoding ◽  
Genomic Dna Library ◽  
Mouse Genomic

The Drosophila segmentation gene Krüppel encodes multiple tandemly repeated units predicted to form DNA-binding zinc fingers. We have isolated 23 bacteriophages, containing nonoverlapping inserts from a mouse genomic DNA library, on the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Krüppel finger region. Nucleotide sequence analysis of six phage DNAs indicated that they all contained regions with similarity to Krüppel and potentially encoded zinc finger domains. Within these regions, the level of similarity to Krüppel was particularly high between successive fingers. Northern (RNA) blotting analysis suggested that the mouse sequences belonged to different genes, the expression of some of which was modulated during cell differentiation and development. Hybridization experiments suggested that the similarity between some of the genes extended outside of the finger regions. In conclusion, our data suggest that the mouse genome contains a large family of evolutionarily related genes encoding possible trans-acting factors. These genes are likely to play a regulatory role at the transcriptional level.

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