Sequence characterization and analysis

Abstract In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking re - gions by chromosome walking and perform an in silicomapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen mo - lecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2-4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence map- ping in silicoin the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different mark- ers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize.

This may reflect HRV-C’s capability to adapt to the higher temperature environment of the lower part of the human respiratory tract and thus differentiate it on some phenotypic level from other HRV species. This finding might also support several epidemiological stu- dies on HRV in that HRV-C was more predominantly found in acute lower RTI cases than HRV-A and HRV- B and may significantly contribute to severe respiratory tract disease development, especially the exacerbation of asthma and wheezing. However, sequence analyses of other picornaviruses such as human hepatitis A viruses, hepatotropic members of the genus Hepatovirus, which replicate primarily in the gastrointestinal tract and spread to the liver causing liver failure and jaundice have shown a much lower G+C content [55]. To further understand this finding and investigate the mechanisms of virus-induced asthma exacerbations, HRV-C’s mode of infection should be further investigated.

ABSTRACT The signal recognition particle (SRP) is a ribonucleoprotein complex whose components are highly conserved throughout all three domains of life, where it functions to target proteins to extracytoplasmic locations. In Escherichia coli, the SRP is comprised of a single essential protein (Ffh) in complex with a 4.5S RNA species. To better understand how the structure of Ffh contributes to its function, we have used genetic approaches to isolate and characterize new ffh mutants altered in two distinct domains of the protein. Both domains of interest have been implicated as being important for binding to hydrophobic signal peptides of membrane proteins. These studies include using a random sequence approach to identify amino acids important for activity of the finger loop domain. The finger loop was identified from structural analysis as a ~20 amino acid domain with the unusual properties of being both hydrophobic and exposed near the surface of Ffh. Approximately 1% of the random sequences were able to replace the FL domain of Ffh. Bioinformatic analysis of the random sequences revealed that all of the complementing sequences followed a trend of high hydrophobicity at the amino-terminus that decreased towards the carboxy end.

i SUMMARY Fish and shellfish are the second largest source of protein for man after meat products and in some countries, such as Japan, constitute the main source of protein. In recent years, indigenous marine bacteria were responsible for 20% of all diseases and 99% of fatalities associated with the consumption of fishery products (Cozzi and Ciccaglioni, 2005). Among these, the main causes of diseases are some species of Vibrionaceae, which can cause gastroenteritis, especially after the consumption of fish products, raw or undercooked, from temperate and warm Seas. Vibrio is a very diverse genus responsible of different human and animal diseases. The accurate identification of Vibrio spp. is very important to assess the risks in regard to public health and diseases of aquatic organisms. Thus, analyses of population structure for a reliable bacteria characterization in different ecological environments are necessary. In particular, sequence based identification methods are preferable over classical biochemical approaches. In this study, a Multilocus Sequence Analysis scheme was developed on the basis of four housekeeping genes (gyrB, pyrH, recA and atpA) applied to 3 set of Vibrio strains (154 isolates from mollusks in 2007; 92 isolates from crustacean and 22 isolates form mollusks in 2011 ) and 29 reference strains. Concatenated sequences were used for phylogenetic and population analyses and the results were compared with biochemical identification tests (Alsina’s scheme). The phylogeny provided a good clustering, showing 15 clusters and 6 single strains in the first set of strains; 10 clusters and 4 singletons in second set; and 4 clusters and 4 singletons in the third set of strains. The population analysis highlighted 17 subpopulations in first set and 12 subpopulations in second set of Vibrio strains that were well supported by phylogeny with few exceptions. Overestimations of risk due to biochemical identification have been found for V. parahaemolyticus and V. vulnificus and no V. cholerae strains were identified. The false negative results of Alsina’s scheme need to be considered as it might represent a potential public health risk. These findings highlight the need of a rapid and robust identification of shellfish associated foodborne Vibrio spp. and, in addition, the connection of environmental information to genetic data could enhance the Vibrio spp. characterization.

Localization of MAb reac- tivity to a region carboxy terminal to aa 235 of the pCD41 ORF, together with the absence of other reading frames in the cDNA sequence, absence of high-molecula[r]

ISSN: 2319-7706 Volume 10 Number 01 (2021) Journal homepage: http://www.ijcmas.com A792 bp fragment of iNOS gene in guinea fowl has been successfully amplified using the primers designed from available sequence of iNOS gene of chicken. This fragment is equivalent to the chicken iNOS in size and no insertion or deletion was observed. Sizable polymorphism was observed between guinea fowl and other poultry species for iNOS nucleotide sequence. The ratio between non-synonymous to synonymous nucleotide substitutions was 1:4.6 between guinea fowl and chicken, whereas it was 1:4.3 between guinea fowl and quail. The iNOS amino acid sequence comprised of 263 amino acids in guinea fowl. The nucleotide sequence alignment of Gf-iNOS gene with the iNOS genes from other poultry species identified 7 SNPs i.e. T/A, C/T, T/C, A/T, G/A, G/A and C/T substitution at 320, 370, 409, 460, 484, 520 and 553 nt positions, respectively in our Gf- iNOS sequence. For Gf-iNOS PCR-RFLP, Mbo I, Sau3A I, Nde II and Dpn I were identified. Similarly, for PCR-RFLP of chicken iNOS, Rsa I, Eco47 III and Hae II; and for quail iNOS, Mn lI, Hpa II, Nci I, Msp I and Nsi I were identified.Based on nucleotide sequence comparisons for iNOS gene, guinea fowl showed very high and almost equal per cent identity (95.6 – 96.2) with chicken and quail. Quail and chicken also showed very high per cent identity (96.7-97.0) between them. phylogenetic analysis also revealed the similar trend. The polymorphism identified in iNOS CDS in present study, may be utilized for exploring the mechanism of higher disease resistance in guinea fowl, which may further be exploited to develop disease resistant chicken population.

The samples were carried to the University of Rome Tor Vergata where the molecular analysis was performed. DNA extraction was per- formed by cutting about 2 cm 2 of the smeared filter paper and using it for the extraction by QIAamp DNA Micro Kit (QIAgen, Gmbh, Hilden, Germany) in according to the manufacturer's recommendations. The final eluate was stored at −20 °C. In order to unequivocally assigned Giardia isolates at the assemblage level and identify sub-assemblages, PCR was carried out to amplify a 130 bp region from the small subunit ribosomal RNA (ssu-rDNA) [8], and a 432 bp region of the glutamate dehydrogenase (gdh) gene [9]. Positive (Giardia DNA) and negative (no template added) control samples were used in all PCR runs. PCR products were separated by electrophoresis in 1% agarose gel and amplicons were puri fied using the NucleoSpin® Extract kit (Macherey- Nagel GmbH & Co. KG, Germany). Both strands were sequenced by the Bio-Fab Research s.r.l. (Rome, Italy). Sequences were edited using the FinchTV 1.4 software (Geospiza, Inc., Seattle, WA). Consensus sequence was determined by alignment of respective forward and reverse se- quences. Assignment to assemblage and sub-assemblage of G. duodenalis isolates was carried out by sequence comparison (ssu-rDNA) and phenetic analysis (gdh). Multiple alignments were performed using ClustalW2 software for DNA against known sequences available in GenBank for Giardia assemblages. Phenetic analysis was performed using the software MEGA5, conducted using the Tamura 3-parameter method and the phenetic tree was constructed by the Neighbor-Joining algorithm. Bootstrap values were calculated by analyzing 1000 repli- cates. All gdh sequences obtained in this study were deposited in the GenBank database and are available under the accession numbers:

2. John, G.H., Smith, R., Abraham, K.J., Ellis, R.P.: Identification and grouping of Dichelobacter nodosus, using PCR and sequence analysis. Mol. Cell Probes. 1999; 13: 61-65. 3. Stewart, D.J., Peterson, J.E., Vaughan, J.A., Clark, B.L., Emery, D.L., Caldwell, J.B., Kortt, A.A.: The pathogenicity and cultural characteristics of virulent, intermediate and benign strains of Bacteroides nodosus causing ovine footrot. Aust. Vet. J., 1986;

Abstract: The aim of this study was to investigate the amount of variation between Acipenseridea family and also, study amount of phylogenetic variation between Persian sturgeon (Acipenser persicus) with other Acipenseridea regarding to the Growth Hormon (GH) gene. In this study, pre growth hormone gene of Persian sturgeon was identified for first time and applied to gene bank (JN604534.1, 2011). The total RNA was extracted from pituitary gland of Persian Sturgeon, cDNA was synthesized. The full-length cDNA sequence of Persian sturgeon contains a 645 nucleotide open reading frame, which encodes a peptide of 214 amino acids. The position of the signal peptide cleavage site was predicted to be at position 72. After cleaving of a signal peptide of 24 amino acid residues and a mature peptide of 190 aa formed. Multiple sequence alignments and Phylogenic tree were performed by using the MEGA5 program. By sequence alignment and phylogenetic analysis of amino acid residues indicated that GH widely conserved in other species were identified. The GH nucleic acid and amino acid residue sequences of Persian sturgeon had a highest similarity to these of other Acipenseridea as well as mammalian, followed by those of Anguiliformes.

The present results obtained from CZE and N-termi- nal sequence analysis clearly show that the latex allergens derive from hevein-related molecules: the allergenic com- po[r]

Correspondence should be addressed to Xingqin Qi, [email protected] Received 4 November 2011; Accepted 26 December 2011 Academic Editors: S. Cacchione and A. Pask Copyright © 2012 Xingqin Qi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sequence comparison is a primary technique for the analysis of DNA sequences. In order to make quantitative comparisons, one devises mathematical descriptors that capture the essence of the base composition and distribution of the sequence. Alignment methods and graphical techniques (where each sequence is represented by a curve in high-dimension Euclidean space) have been used popularly for a long time. In this contribution we will introduce a new nongraphical and nonalignment approach based on

Four of 45 candidate genes that produced PCR products from all isolates and generated the greatest number of alleles were included in MLST-7. These loci, the proteins they encode, and their position within a reference GenBank sequence are as follows: purA, adenylosuccinate synthetase positions 568 to 968 in JF275101; fdh, formate dehydrogenase positions 888 to 1236 in JF275100; ack, acetate kinase positions 1 to 648 in JX501660; and sar, sodium sulfate symporter positions 246 to 683 in JF275103. The primers used to produce the PCR prod- ucts containing these loci and the sizes of the amplicons are listed in Table 2. The three housekeeping genes in MLST-4, tuf, cpn60, and pta, were included in MLST-7 because their useful- ness for population genetic analysis has already been estab- lished and data for these loci have been obtained from numer- ous isolates in several studies (1, 2, 4, 8, 18).

And the phylogenetic analyses showed the same location of CUK-3 strain with the GII-4/2006b cluster in the phylogenetic tree. Conclusions: In This study, a first concerning the full-length sequence of a NoV variant in South Korea is meaningful in that it can be used not only as a full-length NoV variant sequence standard for future comparison studies, but also as useful material for the public health field by enabling the diagnosis, vaccine development, and prediction of new emerging variants.

Figure 4.—Amino acid sequence alignment of predicted Als proteins corresponding to the PCR-amplified region. Amino acid sequences of Als proteins from C. albicans were aligned with those predicted from the PCR-amplified C. dubliniensis and C. tropicalis sequences. A consensus sequence is provided. The positions of conserved and semiconserved Cys residues are double- underlined in the consensus sequence. Amplification of the original clones with the Pfu proofreading polymerase and double- stranded sequencing of each fragment suggested that lack of the last Cys residue in Alst2p was not due to a PCR-induced or DNA sequencing error. Because their gene fragments were amplified with the second primer pair and yielded a shorter product, amino acid sequences of Alst2p and Alst3p do not begin until the third sequence block. Gaps in the alignment are denoted by periods; a tilde (ⵑ) is used to indicate sequence information that lies outside of the PCR-amplified region.

In conclusion, we identified a new wheat virus that is associated with wheat leaf yellowing disease, sequenced, and analyzed its whole genome sequence and organization. Additionally, we constructed an infectious cDNA clone of WLYaV that can systemically infect plants of N. benthamiana and cause leaf yellowing symptoms, thus adding a step to fulfill Koch’s postulates. There are still many questions about WLYaV to be answered: what are its natural vector insects, host ranges, distribution and damage in China? However, we anticipate that findings from this study will lead to a better understanding of the incidence and distribution of different wheat viruses in China, and then determine the best control measures.

The presence of slower migrating heteroduplex DNA We sequenced both products amplified from a single made it possible to detect heterozygotes on the diploid megagametophyte and found the 496 bp product to panel using short gels (10 cm), even when differences be identical to the 440 bp product except that it was in sizes among alleles were small. But, when alleles dif- extended by 56 bp of sequence that was composed of fered in length by less than 10 bp, and for Sb01, long 35 bp of additional 39-UTR sequence (as seen in cDNA gels (22 cm) were necessary to assess allelic segregation SB35) plus the 21 bp of primer SB35-R. There was no among megagametophytes and to assign diploid geno- sequence at this location in cDNA SB35 bearing any types with confidence. Genotypes of Sb21 were the least resemblance to a SB35-R priming site and it is not clear resolved of these 12 loci. Three alleles could be distin- how the alleles differed such that priming also occurred guished in homozygotes or haploid megagametophytes, at this distal site in some genotypes.

full fasta sequence entries from the NCBI-NT database corresponding to the GI numbers were extracted using the fastacmd tool [22]. This allows for longer portions of the entire rRNA cistron region (including ITS1, ITS2, 18S, 5.8S, and 28S genes) to be incorporated in the cus- tom database when available. For the final set of anno- tated sequences, we required sequences not to represent uncultured, environmental, or unclassified taxa. In sev- eral cases, the taxonomic lineages of the reference sequences were incomplete, i.e. they were lacking assign- ments at some of the taxonomic levels (Example: “No class level; Zygophyllales; Krameriaceae; Krameria; Krameria ixine”). In these cases, missing fields in the reference database were filled with, for example, "No_class" or “No_order”. All reference sequences were clustered using a 100% identity threshold to identify identical sequences, which were not removed from the database. The final cus- tom database (named clovr-itsdb version 1.0) consists of 154,050 sequences representing over 8,440 genera and over 60,000 species. The 154,050 sequences belonged to 121,993 clusters that contained between one (107,397 clusters) and 103 identical sequences. In many cases, sequences in a cluster differed in length such that the shorter sequences perfectly matched a subsequence of a longer one. These differences in length can impact the e- values of BLASTN search results used for taxonomic as- signment of ITS amplicons (see below). Of the 14,596 clusters with multiple sequences, 4,966 clusters contained sequences with different annotations, typically at the spe- cies level; 905 showed differences at the genus level. Use of fasta (sequence) and txt (taxonomic assignments) file formats for clovr-itsdb allows for easy database expansion, as additional ITS amplicon sequence data become avail- able, as well as database curation, e.g. in cases where iden- tical reference sequences have different annotations.

criteria. One valuable aspect of the MinION sequencer is its portability and the potential applications for field-based analysis. This has led to the development of the field sequencing kit (ONT), which has the dual advantages of (i) not requiring a cold chain due to lyophilization, and (ii) more rapid processing times than with other available library preparation kits. We found that library preparation was more rapid with the field sequencing kit, requiring only 15 min to generate the libraries after nucleic acid extraction and enrichment. However, the performance of the MinION sequencer with a sample prepared with the field sequencing kit demonstrated longer delays for se- quence generation, with increased latency between available data files. Additionally, sequence data could not be fully resolved from samples processed with the field sequencing kit, in contrast to the rapid library prep kit. Nevertheless, use of the field sequencing kit as a simple library preparation kit, along with the MinION system as a portable real-time sequencer, could be employed in the event of high suspicion of disease and when laboratory facilities or infrastructure are not available. Although the field kit will not provide the same degree of comprehensive genome coverage, the kit

Regulation of viral transcription by the matrix protein of vesic- ular stomatitis virus probed by monoclonal antibodies and temperature-sensitive mutants. Monoclonal antibodies to the M [r]

Reverse transcription coupled with PCR was used for the detection of foot-and-mouth disease virus serotypes A, C, and O in organ extracts from experimentally infected cattle. Primers were selected from conserved sequences flanking the genome region coding for the major antigenic site of the capsid located in the C-terminal part of viral protein 1 (VP1). Because this region of the capsid is highly variable its coding sequence is considered to be the most appropriate for the characterization of virus isolates and, therefore, for the determination of the epidemiological relationships between viruses of the same serotype. For differentiation between serotypes and for detailed characterization of individual virus isolates restriction enzyme cleavage and nucleotide sequence analysis of the respective PCR products were carried out. In order to minimize the time required for sample preparation from clinical material, viral RNA was released from particles by heating the sample for 5 min at 90 & C. Finally, an air thermocycler was used, which allows performance of a PCR of 30 cycles in approximately 20 min. The results show that reverse transcription PCR followed by restriction enzyme analysis and/or nucleotide sequence analysis of the PCR products is useful for the rapid detection and differentiation of foot-and-mouth disease virus.