Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

1986 ◽  
Vol 146 (2) ◽  
pp. 166-169
Author(s):  
C. Sadhu ◽  
S. Dutta ◽  
K. P. Gopinathan
Keyword(s):  
Dna Sequences ◽  
Mycobacterium Smegmatis ◽  
Mycobacteriophage I3

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA

1983 ◽  
Vol 136 (4) ◽  
pp. 275-280 ◽  
Author(s):  
S. Sadashiva Karnik ◽  
K. P. Gopinathan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Mycobacteriophage I3

scholarly journals Mycobacterium smegmatis whmD and its homologue Mycobacterium tuberculosis whiB2 are functionally equivalent

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2735-2747 ◽  
Author(s):  
Tirumalai R. Raghunand ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Dna Sequences ◽  
Mycobacterium Smegmatis ◽  
Essential Gene ◽  
Cell Envelope ◽  
Protein Product ◽  
Polyclonal Antiserum ◽  
C Terminus ◽  
Gene Assay

Mycobacterium smegmatis whmD is is an essential gene involved in cell division. This paper shows that whmD and its homologue whiB2 in Mycobacterium tuberculosis are functionally equivalent. The genes are syntenous, and share significant homology in both their coding and non-coding DNA sequences. Transcription site mapping showed that the two genes possess near-identical promoter elements, and they displayed comparable promoter strengths in a reporter gene assay. The two proteins show near identity in their C-terminus, and polyclonal antiserum to WhmD specifically cross-reacts with a ∼15 kDa band in M. tuberculosis lysates. Following overexpression of sense and anti-sense constructs in their cognate mycobacterial hosts, whiB2 and whmD transformants displayed a small-colony phenotype, exhibited filamentation, and showed a reduction in viability. These observations reveal that the two proteins are functionally homologous and that their intracellular concentration is critical for septation in mycobacteria. Colonies of M. tuberculosis overexpressing whiB2 were spherical and glossy, suggesting a change in composition of the cell envelope. Filaments of the conditionally complemented M. smegmatis whmD mutant were non-acid-fast, also indicating changes in characteristics of surface lipids. M. smegmatis transformants carrying a whmD–gfp fusion showed a diffuse pattern of fluorescence, consistent with the putative role of WhmD as a regulator. These observations strongly suggest that M. tuberculosis whiB2 is an essential gene and its protein product in all likelihood regulates the expression of genes involved in the cell division cascade.

scholarly journals Generation of Useful Insertionally Blocked Sterol Degradation Pathway Mutants of Fast-Growing Mycobacteria and Cloning, Characterization, and Expression of the Terminal Oxygenase of the 3-Ketosteroid 9α-Hydroxylase in Mycobacterium smegmatis mc2155

2006 ◽  
Vol 72 (10) ◽  
pp. 6554-6559 ◽  
Author(s):  
Attila Andor ◽  
Antónia Jekkel ◽  
David A. Hopwood ◽  
Ferenc Jeanplong ◽  
Éva Ilkőy ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Mycobacterium Smegmatis ◽  
Genomic Dna ◽  
Degradation Pathway ◽  
Open Reading Frame ◽  
Temperature Sensitive ◽  
Reading Frame ◽  
Insertional Mutants ◽  
Transposon Insertion ◽  
Mycobacterium Phlei

ABSTRACT Integration of the pCG79 temperature-sensitive plasmid carrying Tn611 was used to generate libraries of mutants with blocked sterol-transforming ability of the sterol-utilizing strains Mycobacterium smegmatis mc2155 and Mycobacterium phlei M51-Ept. Of the 10,000 insertional mutants screened from each library, 4 strains with altered activity of the sterol-degrading enzymes were identified. A blocked 4-androstene-3,17-dione-producing M. phlei mutant transformed sitosterol to 23,24-dinorcholane derivatives that are useful starting materials for corticosteroid syntheses. A recombinant plasmid, pFJ92, was constructed from the genomic DNA of one of the insertional mutants of M. smegmatis, 10A12, which was blocked in 3-ketosteroid 9α-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9α-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9α-hydroxylase in M. smegmatis mc2155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9α position.

Transcription factor/DNA interactions visualized by electron spectroscopic imaging

1992 ◽  
Vol 50 (1) ◽  
pp. 450-451
Author(s):  
David P. Bazett-Jones ◽  
Mark L. Brown
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Phosphorus Content ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Dna Backbone ◽  
Two Parameters ◽  
High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

1990 ◽  
Vol 48 (1) ◽  
pp. 282-283
Author(s):  
José L. Carrascosa ◽  
José M. Valpuesta ◽  
Hisao Fujisawa
Keyword(s):  
Dna Sequences ◽  
Expression Vector ◽  
Three Dimensional ◽  
Deae Cellulose ◽  
Dna Packaging ◽  
Dimensional Structure ◽  
Two Dimensional ◽  
Freezing And Thawing ◽  
Three Dimensional Structure ◽  
Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

DNA methylation in satellite repeats disorders

2019 ◽  
Vol 63 (6) ◽  
pp. 757-771 ◽  
Author(s):  
Claire Francastel ◽  
Frédérique Magdinier
Keyword(s):  
Dna Methylation ◽  
Human Genome ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Satellite Repeats ◽  
Tremendous Progress ◽  
Genes Encoding ◽  
Dna Elements ◽  
Near Future

Abstract Despite the tremendous progress made in recent years in assembling the human genome, tandemly repeated DNA elements remain poorly characterized. These sequences account for the vast majority of methylated sites in the human genome and their methylated state is necessary for this repetitive DNA to function properly and to maintain genome integrity. Furthermore, recent advances highlight the emerging role of these sequences in regulating the functions of the human genome and its variability during evolution, among individuals, or in disease susceptibility. In addition, a number of inherited rare diseases are directly linked to the alteration of some of these repetitive DNA sequences, either through changes in the organization or size of the tandem repeat arrays or through mutations in genes encoding chromatin modifiers involved in the epigenetic regulation of these elements. Although largely overlooked so far in the functional annotation of the human genome, satellite elements play key roles in its architectural and topological organization. This includes functions as boundary elements delimitating functional domains or assembly of repressive nuclear compartments, with local or distal impact on gene expression. Thus, the consideration of satellite repeats organization and their associated epigenetic landmarks, including DNA methylation (DNAme), will become unavoidable in the near future to fully decipher human phenotypes and associated diseases.

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