Identification of mariner-like elements in Sitodiplosis mosellana (Diptera: Cecidomyiidae)

2006 ◽  
Vol 138 (2) ◽  
pp. 138-146 ◽  
Author(s):  
O. Mittapalli ◽  
R.H. Shukle ◽  
I.L. Wise
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Blot Analysis ◽  
Copy Number ◽  
Southern Blot Analysis ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Degenerate Pcr ◽  
Sitodiplosis Mosellana ◽  
Wheat Midge

AbstractMariner-like element sequences were recovered from the genome of the orange wheat midge, Sitodiplosis mosellana (Géhin), with degenerate PCR primers designed to conserved regions of mariner transposases. The deduced amino acid sequences of the mariner-like transposases from S. mosellana showed 67% to 78% identity with the peptide sequences of other mariner transposases. A phylogenetic analysis revealed that the mariner-like elements from S. mosellana grouped in the mauritiana subfamily of mariner transposons. Results from Southern blot analysis suggest mariner-like elements are at a moderate copy number in the genome of S. mosellana.

Primary structure and evolution of the ATP-binding domains of the P-type ATPases in Tetrahymena thermophila

1997 ◽  
Vol 272 (2) ◽  
pp. C715-C728 ◽  
Author(s):  
S. Wang ◽  
K. Takeyasu
Keyword(s):  
Amino Acid ◽  
Southern Blot ◽  
Blot Analysis ◽  
Multigene Family ◽  
Southern Blot Analysis ◽  
Tetrahymena Thermophila ◽  
Amino Acid Sequences ◽  
Ion Pumps ◽  
Atpase Gene ◽  
P Type

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pumps show a high degree of divergence in their primary structures but share a limited number of common amino acid residues for their ATP-catalytic function. Particularly, the amino acid sequences for the phosphorylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligonucleotides corresponding to these regions, we applied a polymerase chain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase genes were identified. Sequence comparisons revealed that seven of them can be compiled into a multigene family, which is similar to animal Na+-K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)plasmic reticulum Ca2+-ATPase gene, and the other four share a significant homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR demonstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of these genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-ATPase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation with the amino acid sequences of the cloned cytoplasmic loop region (between the phosphorylation and the gamma-[4-(N-2-chloroethyl-N-methylamino)]-benzylamido ATP sites), the genes with >80% identity form a cognate linkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylogenetic analysis showed that this multigene family is the result of a series of gene duplications.

Cloning and Characterization of the Zebrafish mll Ortholog.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1249-1249
Author(s):  
Blaine W. Robinson ◽  
Giuseppe Germano ◽  
Yuanquan Song ◽  
Rita J. Balice-Gordon ◽  
Carolyn A. Felix
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Temporal Pattern ◽  
Southern Blot Analysis ◽  
Early Embryogenesis ◽  
Amino Acid Sequences ◽  
Pcr Analysis ◽  
Adult Zebrafish ◽  
Wild Type ◽  
Rt Pcr

Abstract Introduction: Zebrafish enable studies of early embryogenesis and hematopoiesis like no other animal models. Many zebrafish orthologs of hematopoietic genes have been identified, and zebrafish models of leukemia are emerging but the zebrafish ortholog of MLL, a critical oncogene disrupted by leukemogenic translocations, has not yet been studied. We cloned the complete zmll cDNA and characterized its temporal expression as a framework for further studies of MLL where current knowledge is still incomplete. Methods: Bioinformatic tools were employed to interrogate the existence and relationship of a zmll ortholog and synteny with human MLL. Degenerate RT-PCR was used to determine whether MLL amino acid sequences in domains highly conserved across species could identify the orthologous zebrafish transcript. Cross-species Southern blot analysis was performed to determine if a predicted zmll gene from restriction map simulations of a projected genomic sequence could be detected with a human cDNA probe for the MLL breakpoint cluster region (bcr). The full-length zmll cDNA was obtained using a combination of 5′ RACE PCR, long-range and conventional PCR. The corresponding protein was analyzed in a phylogram tree. The temporal pattern of zmll RNA expression was examined using quantitative (Q) RT-PCR. Results: Bioinformatic analysis using the human MLL protein as the reference sequence identified two putative “similar to MLL proteins” and predicted two proximal transcript sequences on zebrafish chromosome 15. Gene prediction tools suggested a single genomic structure matching both protein sequences. A conserved block of synteny containing several linked genes surrounded the predicted zmll. Furthermore, zmll and human MLL were in the same map order in an uninterrupted segment with the gene for ubiquitination factor E4A. Degenerate RT-PCR analysis of wild-type adult zebrafish RNA based on cross-species amino acid sequences from highly conserved PHD and SET domains amplified the predicted transcript. Cross-species Southern blot analysis with the human probe detected the projected zmll genomic fragments corresponding to the MLL bcr in adult zebrafish genomic DNA. RT-PCR analysis of wild-type adult zebrafish RNA determined that the two predicted “similar to MLL proteins” were from a single gene. The full length 12657 bp, 35-exon zmll cDNA cloned from wild-type 24 hpf zebrafish embryo RNA predicted a 4218 amino acid protein with CXXC, PHD, Bromodomain, FYRN, FYRC, and SET domains and taspase cleavage sites with 45.8% sequence identity and 57.3% similarity to human MLL. Phylogram tree analysis suggested evolutionary divergence of mammals from teleosts but nonetheless conservation of critical functional domains. QRT-PCR demonstrated maternally supplied zmll mRNA during the earliest embryonic developmental timepoints, and expression of zygotic zmll mRNA during embryogenesis and in the zebrafish adult. Conclusions: These results indicate that there is a single zmll gene with highly conserved functional similarity to human MLL. The temporal pattern of expression, including maternal supply of transcript to the embryo, indicates that zmll is important from early embryogenesis through the entire lifespan of the fish. The high evolutionary conservation of critical domains creates the framework to use zebrafish for studying MLL in hematopoiesis and leukemia.

Characterization and comparative analysis of three low molecular weight glutenin C-subunit genes isolated from Aegilops tauschii

2007 ◽  
Vol 87 (2) ◽  
pp. 273-280 ◽  
Author(s):  
Yu-He Pei ◽  
Ai-Li Wang ◽  
Xue-Li An ◽  
Xiao-Hui Li ◽  
Yan-Zhen Zhang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Aegilops Tauschii ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Low Molecular Weight ◽  
Primitive Form ◽  
Reading Frame ◽  
Specific Pcr

Three low molecular weight glutenin subunit (LMW-GS) genes from T121, T128 and T132 accessions of Aegilops tauschii (DD, 2n = 2x = 14) were amplified using allelic-specific PCR primers. The amplified products with a size of about 900 bp were cloned and sequenced. Three complete coding sequences of LMW-GS with 918 bp, 921 bp and 918 bp were obtained and named as LMW-T121, LMW-T128, LMW-T132, respectively. Each gene contained a complete open reading frame and had no introns. The deduced amino acid sequences showed that all belonged to LMW-m type subunit with a predicted molecular weight of about 32 kDa, corresponding to the size of LMW C-subunits. All three subunits possessed eight cysteine residues and had greater homology with previously characterized LMW-m subunits from bread wheat and related species than LMW-s or LMW-i sequences. Some amino acid substitutions and insertion/deletion variations among the sequences were detected. The corresponding three C-subunits in seed endosperm encoded by LMW-T121, LMW-T128, LMW-T132, respectively, were identified and confirmed by SDS-PAGE, MALDI-TOF-MS and direct N-terminal amino acid sequencing. Phylogenetic analysis demonstrated that LMW-m and LMW-s type subunit genes possessed higher identity and they were obviously separated from LMW-i type subunit genes. The LMW-m type might be the primitive form while the LMW-s and LMW-i types are variant forms. Key words: Aegilops tauschii, LMW-GS, AS-PCR, phylogenetic analysis

scholarly journals Phylogenetic Analysis: Basic Concepts and Its Use as a Tool for Virology and Molecular Epidemiology

2018 ◽  
Vol 44 (1) ◽  
pp. 20
Author(s):  
Eloiza Teles Caldart ◽  
Helena Mata ◽  
Cláudio Wageck Canal ◽  
Ana Paula Ravazzolo
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Molecular Epidemiology ◽  
Phylogenetic Analyses ◽  
Phylogenetic Reconstruction ◽  
Evolutionary Process ◽  
Amino Acid Sequences ◽  
Evolutionary Models ◽  
Reconstruction Methods ◽  
Basic Concepts

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology.Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. There are a number of evolutionary models available, varying in complexity according to the number of parameters (transition, transversion, GC content, nucleotide position in the codon, among others). Some papers presented herein provide techniques that can be used to choose evolutionary models. After the model is chosen, the next step is to opt for a phylogenetic reconstruction method that best fits the available data and the selected model. Here we present the most common reconstruction methods currently used, describing their principles, advantages and disadvantages. Distance methods, for example, are simpler and faster, however, they do not provide reliable estimations when the sequences are highly divergent. The accuracy of the analysis with probabilistic models (neighbour joining, maximum likelihood and bayesian inference) strongly depends on the adherence of the actual data to the chosen development model. Finally, we also explore topology confidence tests, especially the most used one, the bootstrap. To assist the reader, this review presents figures to explain specific situations discussed in the text and numerous examples of previously published scientific articles in virology that demonstrate the importance of the techniques discussed herein, as well as their judicious use.Conclusion: The DNA sequence is not only a record of phylogeny and divergence times, but also keeps signs of how the evolutionary process has shaped its history and also the elapsed time in the evolutionary process of the population. Analyses of genomic sequences by molecular phylogeny have demonstrated a broad spectrum of applications. It is important to note that for the different available data and different purposes of phylogenies, reconstruction methods and evolutionary models should be wisely chosen. This review provides theoretical basis for the choice of evolutionary models and phylogenetic reconstruction methods best suited to each situation. In addition, it presents examples of diverse applications of molecular phylogeny in virology.

scholarly journals Taxonomic status of Bhanja and Kismayo viruses (family Bunyaviridae)

2012 ◽  
Vol 17 (4) ◽  
pp. 4-8
Author(s):  
A. S Klimentov ◽  
A. P Gmyl ◽  
A. M Butenko ◽  
L. V Gmyl ◽  
O. V Isaeva ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Taxonomic Status ◽  
Amino Acid Sequences ◽  
The Family ◽  
L Segment

The nucleotide sequence of M= (1398 nucleotides and L= (6186 nucleotides) segments of the genome of Bhanja virus and L-segment (1297 nucleotides) of Kismayo virus has been partially determined. Phylogenetic analysis of deduced amino acid sequences showed that these viruses are novel members of the Flebovirus (Phlebovirus) genus in the family Bunyaviridae

scholarly journals Molecular characterization and phylogenetic analysis of NBS-LRR genes in wild relatives of eggplant (Solanum melongena L

2018 ◽  
Author(s):  
Sona. S Dev ◽  
P. Poornima ◽  
Akhil Venu
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Sequence Similarity ◽  
Interleukin 1 ◽  
Preliminary Investigation ◽  
Solanum Melongena ◽  
Wild Relatives ◽  
Amino Acid Sequences ◽  
R Genes ◽  
Multiple Sequence

Eggplantor brinjal (Solanum melongena L.), is highly susceptible to various soil-borne diseases. The extensive use of chemical fungicides to combat these diseases can be minimized by identification of resistance gene analogs (RGAs) in wild species of cultivated plants.In the present study, degenerate PCR primers for the conserved regions ofnucleotide binding site-leucine rich repeat (NBS-LRR) were used to amplify RGAs from wild relatives of eggplant (Black nightshade (Solanum nigrum), Indian nightshade (Solanumviolaceum)and Solanu mincanum) which showed resistance to the bacterial wilt pathogen, Ralstonia solanacearumin the preliminary investigation. The amino acid sequence of the amplicons when compared to each other and to the amino acid sequences of known RGAs deposited in Gen Bank revealed significant sequence similarity. The phylogenetic analysis indicated that they belonged to the toll interleukin-1 receptors (TIR)-NBS-LRR type R-genes. Multiple sequence alignment with other known R genes showed significant homology with P-loop, Kinase 2 and GLPL domains of NBS-LRR class genes. There has been no report on R genes from these wild eggplants and hence the diversity analysis of these novel RGAs can lead to the identification of other novel R genes within the germplasm of different brinjal plants as well as other species of Solanum.

scholarly journals Sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12) from Desulfonispora thiosulfatigenes: purification, properties and primary sequence

2001 ◽  
Vol 357 (2) ◽  
pp. 581-586 ◽  
Author(s):  
Karin DENGER ◽  
Jürgen RUFF ◽  
Ulrike REIN ◽  
Alasdair M. COOK
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Thiamine Pyrophosphate ◽  
Strictly Anaerobic ◽  
Laser Desorption Ionization ◽  
Native Form ◽  
Ionization Time ◽  
Sds Page

The strictly anaerobic bacterium Desulfonispora thiosulfatigenes ferments taurine via sulphoacetaldehyde, which is hydrolysed to acetate and sulphite by sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12). The lyase was expressed at high levels and a two-step, 4.5-fold purification yielded an apparently homogeneous soluble protein, which was presumably a homodimer in its native form; the molecular mass of the subunit was about 61kDa (by SDS/PAGE). The mass was determined to be 63.8kDa by matrix-assisted laser-desorption ionization–time-of-flight (MALDI–TOF) MS. The purified enzyme converted 1mol of sulphoacetaldehyde to 1mol each of sulphite and acetate, but no requirement for thiamine pyrophosphate (TPP) was detected. The N-terminal and two internal amino acid sequences were determined, which allowed us to generate PCR primers. The gene was amplified and sequenced. The DNA sequence had no significant homologue in the databases searched, whereas the derived amino acid sequence indicated an oxo-acid lyase, revealed a TPP-binding site and gave a derived molecular mass of 63.8kDa.

Detection of the Na+-translocating NADH-quinone reductase in marine bacteria using a PCR technique

2000 ◽  
Vol 46 (4) ◽  
pp. 325-332 ◽  
Author(s):  
Sanae Kato ◽  
Isao Yumoto
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
16S Rrna ◽  
Marine Bacteria ◽  
Genomic Dna ◽  
Screening Method ◽  
Quinone Reductase ◽  
Amino Acid Sequences ◽  
Homologous Sequences ◽  
Simple Screening

To examine the distribution of the Na+-translocating NADH-quinone reductase (Na+-NQR) among marine bacteria, we developed a simple screening method for the detection of this enzyme. By reference to the homologous sequences of the Na+-NQR operons from Vibrio alginolyticus and Haemophilus influenzae, a pair of primers was designed for amplification of a part of the sixth ORF (nqr6) of the Na+-NQR operon. When PCR was performed using genomic DNA from 13 marine bacteria, a 0.9-kbp fragment corresponding to nqr6 was amplified in 10 strains. Although there were three PCR-negative strains phylogenetically, based on the sequence of the 16S rRNA, these were placed far from the PCR-positive strains. No product was observed in the case of nonmarine bacteria. The nucleotide and predicted amino acid sequences of nqr6 were highly conserved among the PCR-positive marine bacteria. A phylogenetic analysis of marine bacteria, based on nqr6 sequencing, was performed.Key words: Na+-translocating, NADH-quinone reductase, marine bacteria, PCR.

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