Western, Northern, and Southern Blotting

2008 ◽  
pp. 48-57 ◽  
Author(s):  
Stephan Schlickeiser ◽  
Uwe Pleyer
Keyword(s):  
Southern Blotting

scholarly journals Multiple Chromosomes in Bacteria: The Yin and Yang of trp Gene Localization in Rhodobacter sphaeroides 2.4.1

Genetics ◽  
1999 ◽  
Vol 153 (2) ◽  
pp. 525-538
Author(s):  
Chris Mackenzie ◽  
Adrian E Simmons ◽  
Samuel Kaplan
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Biosynthetic Pathway ◽  
Southern Blotting ◽  
Physical Map ◽  
Functional Integration ◽  
Bacterial Genome ◽  
Single Copy ◽  
Gene Localization ◽  
Yin And Yang ◽  
Trp Genes

Abstract The existence of multiple chromosomes in bacteria has been known for some time. Yet the extent of functional solidarity between different chromosomes remains unknown. To examine this question, we have surveyed the well-described genes of the tryptophan biosynthetic pathway in the multichromosomal photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1. The genome of this organism was mutagenized using Tn5, and strains that were auxotrophic for tryptophan (Trp-) were isolated. Pulsed-field gel mapping indicated that Tn5 insertions in both the large (3 Mb CI) and the small (0.9 Mb CII) chromosomes created a Trp- phenotype. Sequencing the DNA flanking the sites of the Tn5 insertions indicated that the genes trpE-yibQ-trpGDC were at a locus on CI, while genes trpF-aroR-trpB were at locus on CII. Unexpectedly, trpA was not found downstream of trpB. Instead, it was placed on the CI physical map at a locus 1.23 Mb away from trpE-yibQ-trpGDC. To relate the context of the R. sphaeroides trp genes to those of other bacteria, the DNA regions surrounding the trp genes on both chromosomes were sequenced. Of particular significance was the finding that rpsA1, which encodes ribosomal protein S1, and cmkA, which encodes cytidylate monophosphate kinase, were on CII. These genes are considered essential for translation and chromosome replication, respectively. Southern blotting suggested that the trp genes and rpsA1 exist in single copy within the genome. To date, this topological organization of the trp “operon” is unique within a bacterial genome. When taken with the finding that CII encodes essential housekeeping functions, the overall impression is one of close regulatory and functional integration between these chromosomes.

scholarly journals Three Subfamilies of Pheromone and Receptor Genes Generate Multiple B Mating Specificities in the Mushroom Coprinus cinereus

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1115-1123 ◽  
Author(s):  
John R Halsall ◽  
Michael J Milner ◽  
Lorna A Casselton
Keyword(s):  
Southern Blotting ◽  
Transmembrane Domain ◽  
Functional Unit ◽  
Large Family ◽  
Coprinus Cinereus ◽  
Multidrug Transporter ◽  
Type Locus ◽  
Allele Specific ◽  
Unique Specificity ◽  
Major Facilitator

Abstract The B mating type locus of the basidiomycete Coprinus cinereus encodes a large family of lipopeptide pheromones and their seven transmembrane domain receptors. Here we show that the B42 locus, like the previously described B6 locus, derives its unique specificity from nine multiallelic genes that are organized into three subgroups each comprising a receptor and two pheromone genes. We show that the three genes within each group are kept together as a functional unit by being embedded in an allele-specific DNA sequence. Using a combination of sequence analysis, Southern blotting, and DNA-mediated transformation with cloned genes, we demonstrate that different B loci may share alleles of one or two groups of genes. This is consistent with the prediction that the three subgroups of genes are functionally redundant and that it is the different combinations of their alleles that generate the multiple B mating specificities found in nature. The B42 locus was found to contain an additional gene, mfs1, that encodes a putative multidrug transporter belonging to the major facilitator family. In strains with other B mating specificities, this gene, whose functional significance was not established, lies in a region of shared homology flanking the B locus.

Porcine Repeat Element DNA: In Situ Detection of Xenotransplanted Cells

1995 ◽  
Vol 4 (2) ◽  
pp. 253-256 ◽  
Author(s):  
Henry F. Oettinger ◽  
Amelie Rodrigue-Way ◽  
Joyce J. Bousquet ◽  
Albert S.B. Edge
Keyword(s):  
Southern Blotting ◽  
Host Tissue ◽  
Repeat Element ◽  
Tissue Analysis ◽  
Specific Staining ◽  
Tetrazolium Chloride ◽  
Tissue Sections ◽  
Blue Tetrazolium Chloride ◽  
In Situ Detection

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated anti-digoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/ NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Applications of this technique include detection of transplanted cells or organs across a variety of xenogeneic barriers.

Host–parasite genome relationships in Acridid grasshoppers. II. Patterns of variation in the plasmids of gut bacteria of Caledia captiva and Locusta migratoria

Genome ◽  
1987 ◽  
Vol 29 (2) ◽  
pp. 264-271 ◽  
Author(s):  
D. J. Colgan ◽  
D. A. Willcocks
Keyword(s):  
Southern Blotting ◽  
Locusta Migratoria ◽  
Sequence Diversity ◽  
Extrachromosomal Elements ◽  
Size Uniformity ◽  
Restriction Fragment Pattern ◽  
High Level ◽  
Host Parasite ◽  
Plasmid Diversity ◽  
Very High

Plasmid preparations were made from 110 isolates of Enterobacter cloacae taken from the guts of members of the Caledia captiva complex of grasshoppers to ascertain whether a relationship exists between these extrachromosomal elements and taxonomic variation in the grasshoppers themselves. Fifty-two plasmids, distinguishable by mobility or restriction fragment pattern differences, were identified. Thirty-seven of these were similar in size. Five plasmids were nick translated and used to probe Southern blots. Only three instances of cross homology with another plasmid were found, implying a very high level of sequence diversity in the samples. No explanation of the size uniformity and sequence diversity of the plasmids is entirely satisfactory but it appears most likely that the variation is maintained to serve a variety of adaptive functions. No plasmid was found in grasshoppers of more than one taxon of C. captiva. This may be due to geographical limitations on the distribution of plasmids. If this is so, it remains possible that there is an association of one or more plasmids with taxonomic divergence in this grasshopper complex. Plasmid preparations were also made from 68 bacterial isolates (predominantly E. aerogenes) from laboratory-reared Locusta migratoria and from 72 isolates from other acridid grasshoppers. Plasmids of the size general in C. captiva were discovered in most of these isolates. Some smaller plasmids were also found. As judged by restriction endonuclease digests and Southern blotting, plasmid diversity is much less in this sample of L. migratoria bacteria than in the field-collected C. captiva. The plasmids reported in this paper may be considered as possible vectors for use in the genetic control of locusts. Key words: host–parasite, plasmids, grasshoppers, Enterobacter.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron

Parasitology ◽  
2002 ◽  
Vol 124 (2) ◽  
pp. 177-184 ◽  
Author(s):  
M. B. A. MENDONÇA ◽  
N. S. NEHME ◽  
S. S. SANTOS ◽  
E. CUPOLILLO ◽  
N. VARGAS ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Ribosomal Rna ◽  
Southern Blotting ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Pcr Products ◽  
Phylogenetic Lineages ◽  
Definition Of ◽  
Ribosomal Cistron ◽  
Panstrongylus Geniculatus

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3–Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Sα rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5·8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Sα rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.

scholarly journals Genetic variants of human red-cell membrane sialoglycoprotein β. Study of the alterations occurring in the sialoglycoprotein-β gene

1988 ◽  
Vol 250 (2) ◽  
pp. 407-414 ◽  
Author(s):  
M J Tanner ◽  
S High ◽  
P G Martin ◽  
D J Anstee ◽  
P A Judson ◽  
...  
Keyword(s):  
Genetic Variants ◽  
Southern Blotting ◽  
Protein Product ◽  
Red Cells ◽  
The Other ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Initiation Of Translation ◽  
Beta Gene ◽  
Human Red Cell Membrane

We have studied the DNA of individuals who express an altered sialoglycoprotein beta on their red cells by using Southern blotting with sialoglycoprotein-beta cDNA probes. Individuals of the Leach phenotype do not express any beta (sialoglycoprotein beta) or gamma (sialoglycoprotein gamma) on their red cells, and we show that about 7 kb of DNA, including the 3′ end of the beta gene, is deleted in this DNA. Any protein product of this gene is likely to lack the membrane-associating domain of beta. We have also examined the DNA of two types of other individuals (Yus-type and Gerbich-type) who have red cells that lack beta and gamma, but contain abnormal sialoglycoproteins related to beta. These two types of DNA contain different internal deletions of about 6 kb in the beta gene. We suggest that these deletions result from the presence of two different sets of internal homology in the beta gene, and on this basis we propose structures for the abnormal Yus-type and Gerbich-type sialoglycoproteins which are consistent with the other evidence that is available. We provide evidence that beta and gamma are products of the same gene and suggest a possible mechanism for the origin of gamma based on leaky initiation of translation of beta mRNA.

scholarly journals Genomic Characterization of A Multidrug-resistant Citrobacter freundii Strain ZT01-0079 Co-Producing blaNDM-1 and blaSHV-12 using MiSeq and MinION

2020 ◽  
Author(s):  
Tingyan Zhang ◽  
Yanfeng Lin ◽  
Zhonghong Li ◽  
Xiong Liu ◽  
Jinhui Li ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Southern Blotting ◽  
Multiple Drug Resistance ◽  
Carbapenem Resistance ◽  
Multidrug Resistant ◽  
Multiple Drug ◽  
Citrobacter Freundii ◽  
Rapid Spread ◽  
Genomic Characterization

Abstract Background: The emergence of multi-drug resistant Citrobacter freundii poses daunting challenges to the treatment of clinical infections. The purpose of this study was to characterize the genome of a C. freundii strain with an IncX3 plasmid encoding both the blaNDM-1 and blaSHV-12 genes.Methods: Strain ZT01-0079 was isolated from a clinical urine sample. The Vitek2 system was used for identification and antimicrobial susceptibility testing. The presence of blaNDM-1 was detected by PCR and sequencing. Conjugation experiments and Southern blotting were performed to determine the transferability of the blaNDM-1- carrying plasmid. Nanopore and Illumina sequencing were performed to better understand the genomic characteristics of the strain.Results: Strain ZT01-0079 was identified as C. freundii, and the coexistence of blaNDM-1 and multiple drug resistance genes was confirmed. Electrophoresis and Southern blotting showed that blaNDM-1 was located on a ~53kb IncX3 plasmid. The NDM-1-encoding plasmid was successfully transferred at a frequency of 1.68×10−3. Both blaNDM-1 and blaSHV-12 were located on the self-transferable IncX3 plasmid.Conclusion: The rapid spread of the IncX3 plasmid highlights the importance of continuous monitoring of the prevalence of NDM-1-encoding Enterobacteriaceae. Mutations of existing carbapenem resistance genes will bring formidable challenges to clinical treatment.

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