scholarly journals Transcriptional Organization and Regulation of Magnetosome Operons in Magnetospirillum gryphiswaldense

2006 ◽  
Vol 72 (9) ◽  
pp. 5757-5765 ◽  
Author(s):  
Sabrina Sch�bbe ◽  
Chris W�rdemann ◽  
J�rg Peplies ◽  
Udo Heyen ◽  
Cathrin Wawer ◽  
...  
Keyword(s):  
Sufficient Conditions ◽  
Growth Conditions ◽  
Iron Limitation ◽  
Rt Pcr ◽  
Recognition Sequence ◽  
Magnetospirillum Gryphiswaldense ◽  
Reverse Transcription Pcr ◽  
Intergenic Regions ◽  
Magnetite Biomineralization ◽  
Polycistronic Transcripts

ABSTRACT Genes involved in magnetite biomineralization are clustered within the genomic magnetosome island of Magnetospirillum gryphiswaldense. Their transcriptional organization and regulation were studied by several approaches. Cotranscription of genes within the mamAB, mamDC, and mms clusters was demonstrated by reverse transcription-PCR (RT-PCR) of intergenic regions, indicating the presence of long polycistronic transcripts extending over more than 16 kb. The transcription start points of the mamAB, mamDC, and mms operons were mapped at 22 bp, 52 bp, and 58 bp upstream of the first genes of the operons, respectively. Identified −10 and −35 boxes of the P mamAB , P mamDC , and P mms promoters showed high similarity to the canonical σ70 recognition sequence. The transcription of magnetosome genes was further studied in response to iron and oxygen. Transcripts of magnetosome genes were detected by RT-PCR both in magnetic cells grown microaerobically under iron-sufficient conditions and in nonmagnetic cells grown either aerobically or with iron limitation. The presence of transcripts was found to be independent of the growth phase. Further results from partial RNA microarrays targeting the putative magnetosome transcriptome of M. gryphiswaldense and real-time RT-PCR experiments indicated differences in expression levels depending on growth conditions. The expression of the mam and mms genes was down-regulated in nonmagnetic cells under iron limitation and, to a lesser extent, during aerobic growth compared to that in magnetite-forming cells grown microaerobically under iron-sufficient conditions.

scholarly journals The Chemolithoautotroph Acidithiobacillus ferrooxidans Can Survive under Phosphate-Limiting Conditions by Expressing a C-P Lyase Operon That Allows It To Grow on Phosphonates

2008 ◽  
Vol 74 (6) ◽  
pp. 1829-1835 ◽  
Author(s):  
Mario Vera ◽  
Fernando Pagliai ◽  
Nicolas Guiliani ◽  
Carlos A. Jerez
Keyword(s):  
Acidithiobacillus Ferrooxidans ◽  
Primary Source ◽  
Bioinformatic Analysis ◽  
Growth Conditions ◽  
Rt Pcr ◽  
Inorganic Polyphosphate ◽  
Reverse Transcription Pcr ◽  
Bacterial Enzyme ◽  
Phosphorus Source ◽  
Dna Macroarrays

ABSTRACT The chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans is of great importance in biomining operations. During the bioleaching of ores, microorganisms are subjected to a variety of environmental stresses and to the limitations of some nutrients, such as inorganic phosphate (Pi), which is an essential component for all living cells. Although the primary source of phosphorus for microorganisms is Pi, some bacteria are also able to metabolize Pi esters (with a C-O-P bond) and phosphonates (with a very inert C-P bond). By using bioinformatic analysis of genomic sequences of the type strain of A. ferrooxidans (ATCC 23270), we found that as part of a Pho regulon, this bacterium has a complete gene cluster encoding C-P lyase, which is the main bacterial enzyme involved in phosphonate (Pn) degradation in other microorganisms. A. ferrooxidans was able to grow in the presence of methyl-Pn or ethyl-Pn as an alternative phosphorus source. Under these growth conditions, a great reduction in inorganic polyphosphate (polyP) levels was seen compared with the level for cells grown in the presence of Pi. By means of reverse transcription-PCR (RT-PCR), DNA macroarrays, and real-time RT-PCR experiments, it was found that A. ferrooxidans phn genes were cotranscribed and their expression was induced when the microorganism was grown in methyl-Pn as the only phosphorus source. This is the first report of phosphonate utilization in a chemolithoautotrophic microorganism. The existence of a functional C-P lyase system is a clear advantage for the survival under Pi limitation, a condition that may greatly affect the bioleaching of ores.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

scholarly journals Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico

2020 ◽  
Vol 110 (1) ◽  
pp. 106-120 ◽  
Author(s):  
Avijit Roy ◽  
Andrew L. Stone ◽  
Gabriel Otero-Colina ◽  
Gang Wei ◽  
Ronald H. Brlansky ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sensu Stricto ◽  
Genome Segment ◽  
Rt Pcr ◽  
Sequence Comparisons ◽  
Orchid Fleck Virus ◽  
Reverse Transcription Pcr ◽  
Sequencing Technologies ◽  
Negative Sense

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit–specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit–specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

scholarly journals Novel manifestations of Warburg micro syndrome type 1 caused by a new splicing variant of RAB3GAP1: a case report

BMC Neurology ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Raziyeh Khalesi ◽  
Ehsan Razmara ◽  
Golareh Asgaritarghi ◽  
Ali Reza Tavasoli ◽  
Yasser Riazalhosseini ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Cerebral White Matter ◽  
Global Developmental Delay ◽  
Spastic Diplegia ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Mutation System ◽  
Warburg Micro Syndrome

Abstract Background The present study aimed to determine the underlying genetic factors causing the possible Warburg micro syndrome (WARBM) phenotype in two Iranian patients. Case presentation A 5-year-old female and a 4.5-year-old male were referred due to microcephaly, global developmental delay, and dysmorphic features. After doing neuroimaging and clinical examinations, due to the heterogeneity of neurodevelopmental disorders, we subjected 7 family members to whole-exome sequencing. Three candidate variants were confirmed by Sanger sequencing and allele frequency of each variant was also determined in 300 healthy ethnically matched people using the tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. To show the splicing effects, reverse transcription-PCR (RT-PCR) and RT-qPCR were performed, followed by Sanger sequencing. A novel homozygous variant—NM_012233.2: c.151-5 T > G; p.(Gly51IlefsTer15)—in the RAB3GAP1 gene was identified as the most likely disease-causing variant. RT-PCR/RT-qPCR showed that this variant can activate a cryptic site of splicing in intron 3, changing the splicing and gene expression processes. We also identified some novel manifestations in association with WARBM type 1 to touch upon abnormal philtrum, prominent antitragus, downturned corners of the mouth, malaligned teeth, scrotal hypoplasia, low anterior hairline, hypertrichosis of upper back, spastic diplegia to quadriplegia, and cerebral white matter signal changes. Conclusions Due to the common phenotypes between WARBMs and Martsolf syndrome (MIM: 212720), we suggest using the “RABopathies” term that can in turn cover a broad range of manifestations. This study can per se increase the genotype-phenotype spectrum of WARBM type 1.

scholarly journals Direct infection of CD34+ progenitor cells by human cytomegalovirus: evidence for inhibition of hematopoiesis and viral replication

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1277-1283 ◽  
Author(s):  
M Movassagh ◽  
J Gozlan ◽  
B Senechal ◽  
C Baillou ◽  
JC Petit ◽  
...  
Keyword(s):  
Viral Replication ◽  
Human Cytomegalovirus ◽  
Laboratory Strain ◽  
Inhibitory Effect ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Differentiated Cells ◽  
Cd34 Antigen ◽  
Liquid Suspension ◽  
Infected Cells

We successfully infected fluorescence-activated cell-sorted CD34+ cells from normal cord blood by the human cytomegalovirus (HCMV) laboratory strain Towne. An inhibitory effect of HCMV on clonogenic myeloid progenitors was observed in primary methylcellulose cultures. After an initial 7-day liquid culture of CD34(+)-infected cells, this inhibition was further amplified in secondary methylcellulose cultures, then involving both the myeloid and erythroid lineages. Under these conditions, viral DNA was detected both in erythroid and myeloid colonies using the polymerase chain reaction (PCR), but reverse transcription PCR (RT-PCR) failed to detect viral RNA. In contrast, when CD34(+)-infected cells were maintained in liquid suspension, both immediate, early, and late transcripts were detected as soon as day 3. In addition, viral production was demonstrated in the culture supernatants, thus confirming that a complete viral cycle occurred under liquid conditions. Furthermore, by resorting cells into CD34+ and CD34- fractions, we showed by RT-PCR that viral replication took place in cells still expressing CD34 antigen, whereas no RNA was found in more differentiated cells that had subsequently lost their CD34 antigen. These findings suggest that HCMV replication can occur at the early steps of progenitor differentiation and may be involved in the viral-induced myelosuppression.

Immunological and molecular detection of rotavirus genotype in calves with gastroenteritis in Diyala-Iraq

2021 ◽  
Vol 1 (1) ◽  
pp. 001-013
Author(s):  
Ammar Talib Nasser ◽  
Abdulrazak Shafiq Hasan ◽  
Amer Khazaal Saleh ◽  
Mohammad Kassem Saleh
Keyword(s):  
Molecular Detection ◽  
Cross Sectional Study ◽  
Sectional Study ◽  
Rt Pcr ◽  
Cross Sectional ◽  
Reverse Transcription Pcr ◽  
Group A ◽  
Stool Antigen ◽  
Stool Samples ◽  
Age Range

Aim: To explore the prevalence of rotavirus infection along with the molecular detection and genotyping of group A rotavirus (RVA) among bovine calves up to 5 months old in Diyala province-Iraq. Methods: This is a cross sectional study conducted in Diyala province-Iraq during the period of 2019-2020. One hundred bovine calves with age range of 1-5 months were included in the study. All were suffering acute gastroenteritis. Serum anti-rotavirus IgM and IgG plus fecal rotavirus Ag were tested for using ELISA techniques. Stool samples positive for rotavirus Ag were submitted for reverse transcription PCR (RT-PCR) for G and P genes, followed by sequencing and genotyping thereafter. Statistical analysis was done using SPSS version 25 and P values ≤ 0.05 were considered significant. Results: The positivity rate of anti-rotavirus IgM was 80% (P = 0.0001), and that of anti-rotavirus IgG was 79% (P = 0.0001). The rotavirus stool antigen was detected in 68% of calves (P = 0.01). A total of 45 stool samples which were positive for rotavirus Ag were submitted for RT-PCR; 13 (28.9%) were positive and 32 (71.1%) were negative (P = 0.084). 10 PCR positive samples were used for sequencing and genotyping and indicated that all investigated strains belonged to G1P[8] genotype. Conclusion: The current strains analyzed belonged to the G1P[8] RVA genotypes, affirming that employment of VP7 gene polymorphism accurately yielded uniform phylogenetic distances amongst investigated rotavirus strains and that there were no noticeable assortment events between human and animal rotavirus strains in Diyala province.

Exons sequencing identifies a novel mutation in GPR157 in a high myopia family

2020 ◽  
Author(s):  
Tao Tang ◽  
Mohan Liu ◽  
Ting Wei ◽  
Lin Deng ◽  
Yueyang Zhang ◽  
...  
Keyword(s):  
Nonsense Mutation ◽  
High Myopia ◽  
Sanger Sequencing ◽  
Novel Mutation ◽  
Rt Pcr ◽  
Genetic Characteristics ◽  
Reverse Transcription Pcr ◽  
Whole Exome ◽  
Reduced Penetrance ◽  
Next Generation Sequencing Ngs

Abstract Background Next-generation sequencing (NGS) and whole exome sequencing (WES) have identified many potential disease-causing loci and genetic mutations of high myopia(HM). However, these known genes can only explain the heritability of a small proportion of HM patients. A large proportion of variants have yet to be discovered. Herein we aimed to investigate the genetic characteristics of HM through a Chinese HM family(the inheritance pattern unknown) . Methods We performed WES on the parent-offspring trio and identified mutations by Sanger sequencing. All the members in this family were sequenced to validate phenotype co-segregated with candidate genes via Sanger sequencing as well. Besides, mutations detected were further evaluated in a cohort of 110 sporadic high myopia controls and 200 unrelated ethically-matched controls. And reverse transcription PCR(RT-PCR) was applied to measure the mRNA expression levels of GPR157 in the 4-week-old KM mice. Results A novel heterozygous nonsense mutation, c.859C>T (p.Arg287*) of GPR157 gene, was detected in the proband and her father by WES. And this disease-associated mutation was not found in 310 control individuals. For the family under study, HM was classified as autosomal dominant inheritance with reduced penetrance. And RT-PCR results showed GPR157 was abundantly expressed in the eye. Conclusion The hybrid nonsense mutation of the GPR157 gene identified in this study may constitute a novel genetic cause of HM. Keywords :high myopia, WES, GPR157

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