Cloning and sequencing of the ompL37 gene present in Leptospira interrogans, a surface protein in pathogenic leptospires

Background and Objectives: Leptospirosis, an infection caused by pathogenic leptospires, is associated with insufficient sanitation and poverty. Leptospira is transmitted through contact with contaminated urine of reservoir animals. The primary objective of this study was to clone and sequence the ompL37 gene present in local and vaccine serovars. Materials and Methods: A total of 16 Leptospira interrogans serovars were cultured in EMJH liquid medium. After grow- ing, genomic DNA was extracted using phenol-chloroform method. Primer pair was synthesized to amplify the 996 bp ompL37 sequence. The amplified ompL37 gene was cloned into pTZ57R/T vector. The sequences obtained from this study were compared with an only recorded sequence in the Genbank by the Meg Align software. Results: PCR products showed an amplified 996bp ompL37 gene product belonging to pathogenic serovars, while no ompL37 products were amplified in non-pathogenic serovars. Sequences comparison tests from 16 native serotypes exam- ined in this study displayed a similarity range of 84% to 99.5% among serovars used. The results showed that two serotypes of L. interrogans including Serjoehardjo (RTCC2810 and RTCC2821) had the highest identity up to 95.5%. Two serovars of L. interrogans including Pomona (RTCC2822) and Icterohaemorrhagiae (RTCC2823) had the lowest identity about 84%. Conclusion: As the results showed, ompL37, present on the surface of such bacteria, showed a conserved sequence. ompL37, as a key role in cell adhesion and pathogenicity, can be used for designing diagnostic tests and vaccines. Furthermore, se- quencing of various sites in ompL37 gene, including binding sites and immunogenic epitopes, can be valuable alternatives for future studies.

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Influence of Selective Agents (EMJH-STAFF), Sample Filtration and pH on Leptospira Interrogans Serovar Icterohaemorrhagiae Cultivation and Isolation from Swine Urine

Veterinary Sciences ◽

10.3390/vetsci8060090 ◽

2021 ◽

Vol 8 (6) ◽

pp. 90

Author(s):

Romana Steinparzer ◽

Tamara Mair ◽

Christine Unterweger ◽

Adi Steinrigl ◽

Friedrich Schmoll

Keyword(s):

Diagnostic Tests ◽

Urine Samples ◽

Epidemiological Surveillance ◽

Leptospira Interrogans ◽

Sample Storage ◽

Clinical Specimens ◽

Negative Results ◽

Swine Urine ◽

Selective Agents ◽

Phosphate Buffered Saline

Leptospira spp. cause the zoonotic disease leptospirosis, which occurs in numerous mammalians worldwide. Isolation is still important for serotyping and genotyping of Leptospira, which in turn is essential for epidemiological surveillance of leptospirosis and the development of diagnostic tests and vaccines. However, isolation of Leptospira from clinical specimens is inherently insensitive. This study was conducted to examine the influence of selective agents, sample filtration, sample pH and the use of phosphate buffered saline (PBS) buffer for sample storage to improve the success of cultivation and isolation of Leptospira interrogans serovar Icterohaemorrhagiae from swine urine. EMJH (Ellinghausen McCullough, Johnson and Harris) medium including the selective agents sulfamethoxazole, trimethoprim, amphotericin, fosfomycin and 5-fluorouracil (STAFF) increased the success of Leptospira isolation from spiked swine urine samples. Sample filtration yielded only negative results. Isolation in EMJH-STAFF was successful from swine urine with a density as low as 104 Leptospira/mL, and urine with pH ≤ 7 impaired the cultivation rate. Cultivation and isolation were not improved by the addition of PBS to spiked urine samples prior to storage for 24 h at 4 °C. The results of the study demonstrate that cultivation and isolation of leptospires from swine urine can be improved by enhanced methods.

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A Mutation in Paramecium tetraurelia Reveals Functional and Structural Features of Developmentally Excised DNA Elements

Genetics ◽

10.1093/genetics/148.1.139 ◽

1998 ◽

Vol 148 (1) ◽

pp. 139-149 ◽

Cited By ~ 4

Author(s):

Kimberly M Mayer ◽

Kazuyuki Mikami ◽

James D Forney

Keyword(s):

Mutant Cell ◽

Consensus Sequence ◽

Surface Protein ◽

Structural Features ◽

Inverted Terminal Repeat ◽

Paramecium Tetraurelia ◽

Coding Region ◽

Conserved Sequence ◽

Variable Surface ◽

Dna Elements

Abstract The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28–882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tc1 transposons (Klobutcher and Herrick 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES2591 has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its wild-type micronuclear copy through multiple sexual generations.

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Deciphering the Ligand-binding Sites in theBorrelia burgdorferiComplement Regulator-acquiring Surface Protein 2 Required for Interactions with the Human Immune Regulators Factor H and Factor H-like Protein 1

Journal of Biological Chemistry ◽

10.1074/jbc.m805844200 ◽

2008 ◽

Vol 283 (50) ◽

pp. 34855-34863 ◽

Cited By ~ 37

Author(s):

Corinna Siegel ◽

Johanna Schreiber ◽

Katrin Haupt ◽

Christine Skerka ◽

Volker Brade ◽

...

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Surface Protein ◽

Factor H ◽

Ligand Binding Sites ◽

Human Immune

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Identification of restriction enzyme in the FSHR gene of indonesian local cattle

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/888/1/012024 ◽

2021 ◽

Vol 888 (1) ◽

pp. 012024

Author(s):

P W Prihandini ◽

A Primasari ◽

M Luthfi ◽

D Pamungkas ◽

A P Z N L Sari ◽

...

Keyword(s):

Restriction Enzyme ◽

Restriction Site ◽

Follicle Stimulating Hormone ◽

Restriction Enzymes ◽

Future Studies ◽

Fshr Gene ◽

Pcr Rflp ◽

Mapping Analysis ◽

Pcr Products ◽

Enzyme Mapping

Abstract The restriction enzyme is important for genotyping using the PCR-RFLP technique. Therefore, this study aims to identify the restriction enzyme mapping in the partial sequence of the follicle-stimulating hormone receptor (FSHR) gene in Indonesian local cattle. A total of 29 samples sized 306 bp, were aligned with Genbank sequence acc no. NC_032660, resulting three polymorphic sites, namely g.193G>C, g.227T>C, and g.275A>C. Furthermore, the restriction mapping analysis using the NEBcutter program V2.0 showed that no enzyme recognized the SNP g.275A>C, while the SNP g.193G>C and g.227T>C were identified by the AluI and MscI enzymes, respectively. The AluI enzyme cuts at two positions (193 bp and 243 bp) in the G allele sample producing three fragments namely 50 bp, 63 bp, and 193 bp, meanwhile, in the C allele, the AluI cuts only in position 243 bp, hence, the fragment products are 63 bp and 243 bp. In contrast, the MscI enzyme was only recognized in the T allele, producing fragments sized 77 bp and 229 bp but failed to identify the restriction site along with the PCR products in the C allele. Based on the results, the SNPs (g.193G>C and g.227T>C) and restriction enzymes (AluI and MscI) are applicable for genotyping local Indonesian cattle using the PCR-RFLP technique in future studies.

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Conservation of regulatory elements controlling hairy pair-rule stripe formation

Development ◽

10.1242/dev.117.2.585 ◽

1993 ◽

Vol 117 (2) ◽

pp. 585-596 ◽

Cited By ~ 4

Author(s):

J.A. Langeland ◽

S.B. Carroll

Keyword(s):

Binding Sites ◽

Regulatory System ◽

Regulatory Elements ◽

Drosophila Virilis ◽

Control Individual ◽

Comparative Approach ◽

Conserved Sequence ◽

H Gene ◽

Molecular Comparison ◽

Pair Rule

The hairy (h) gene is one of two pair-rule loci whose striped expression is directly regulated by combinations of gap proteins acting through discrete upstream regulatory fragments, which span several kilobases. We have undertaken a comparative study of the molecular biology of h pair-rule expression in order to identify conserved elements in this complex regulatory system, which should provide important clues concerning the mechanism of stripe formation. A molecular comparison of the h locus in Drosophila virilis and Drosophila melanogaster reveals a conserved overall arrangement of the upstream regulatory elements that control individual pair-rule stripes. We demonstrate that upstream fragments from D. virilis will direct the proper expression of stripes in D. melanogaster, indicating that these are true functional homologs of the stripe-producing D. melanogaster regulatory elements, and that the network of trans-acting proteins that act upon these regulatory elements is highly conserved. We also demonstrate that the spatial relationships between specific h stripes and selected gap proteins are highly conserved. We find several tracts of extensive nucleotide sequence conservation within homologous stripe-specific regulatory fragments, which have facilitated the identification of functional subelements within the D. melanogaster regulatory fragment for h stripe 5. Some of the conserved nucleotide tracts within this regulatory fragment contain consensus binding sites for potential trans-regulatory (gap and other) proteins, while many appear devoid of known binding sites. This comparative approach, coupled with the analysis of reporter gene expression in gap mutant embryos suggests that the Kr and gt proteins establish the anterior and posterior borders of h stripe 5, respectively, through spatial repression. Other, as yet unidentified, proteins are certain to play a role in stripe activation, presumably acting through other conserved sequence tracts.

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Structural organization of upstream exons and distribution of transcription start sites in the chicken c-myb gene

Molecular and Cellular Biology ◽

10.1128/mcb.9.2.837-843.1989 ◽

1989 ◽

Vol 9 (2) ◽

pp. 837-843

Author(s):

S L Hahn ◽

M Hahn ◽

W S Hayward

Keyword(s):

Binding Sites ◽

Noncoding Region ◽

Open Reading Frames ◽

Transcription Start ◽

S1 Nuclease ◽

Transcription Start Sites ◽

Conserved Sequence ◽

Myb Gene ◽

Putative Transcription Factor ◽

Myb Genes

We mapped and sequenced three upstream exons of the chicken c-myb gene and the regions flanking the first coding exon. We found multiple potential binding sites for transcription factors in the 5'-noncoding region, a T-rich stretch of 78 base pairs (bp) (68% T) in the first intron, and four fairly long open reading frames in the antisense direction of the first coding exon and its flanking regions. Three major transcription start sites, contained within a single 11-bp region, were identified by S1 nuclease analysis and primer extension. A sequence comparison of the avian and murine c-myb genes revealed a highly conserved sequence of 124 bp in the 5'-noncoding region. Its location between the putative transcription factor binding sites and the major transcription start sites suggests that it may play an important regulatory role in c-myb expression.

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Sedation Protocol During Bevacizumab Intravitreal Injection in Preterm Infants With Retinopathy of Prematurity

The Journal of Pediatric Pharmacology and Therapeutics ◽

10.5863/1551-6776-23.1.34 ◽

2018 ◽

Vol 23 (1) ◽

pp. 34-40

Author(s):

Jamie L. Miller ◽

Peter N. Johnson ◽

Kari Harkey ◽

R. Michael Siatkowski

Keyword(s):

Retrospective Study ◽

Data Collection ◽

Adverse Events ◽

Retinopathy Of Prematurity ◽

Intravitreal Bevacizumab ◽

Primary Objective ◽

Future Studies ◽

Sedation Protocol ◽

Number Of Patients ◽

Sedation And Analgesia

OBJECTIVES This study describes outcomes of intravenous (IV) analgesics and sedatives for bedside intravitreal bevacizumab injections for retinopathy of prematurity. METHODS This retrospective study included infants receiving intravitreal bevacizumab injections between January 2012 and May 2016. Infants were excluded if bevacizumab was administered under general anesthesia or for incomplete records. Data collection included demographics, sedation and analgesia regimen, and cardiopulmonary adverse events (AEs). The primary objective was to identify the median doses of the IV analgesics and sedatives. The secondary objectives included the number of patients with cardiopulmonary AEs and those with procedure success, defined as procedure completion without interruption and absence of interventions. RESULTS Fifteen infants were included. Fourteen (93.3%) were initiated on a fentanyl infusion at a median of 2 mcg/kg/hr (IQR, 2–3.6), and 12 (80%) received midazolam infusions at a median of 0.06 mg/kg/hr. All patients received at least 1 IV neuromuscular blocker dose just prior to the procedure. Only 2 patients (13.3%) required an increase in their fentanyl or midazolam infusions. Procedure success was achieved in 13 patients (86.7%). Five patients (33.3%) experienced 1 cardiopulmonary AE. One patient (6.7%) had a delay in the procedure, and 1 patient (6.7%) required naloxone. Despite this, the procedure was completed in all patients. CONCLUSIONS Most received fentanyl and midazolam infusions with a dose of vecuronium just prior to the procedure. Thirteen (86.7%) met the criteria for procedure success. One-third experienced a cardiopulmonary AE. Future studies are needed to identify the optimal agents and route of administration for this procedure.

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RAPD-based analysis of differences between male and female genotypes of Asparagus officinalis

Horticultural Science ◽

10.17221/70/2011-hortsci ◽

2012 ◽

Vol 39 (No. 1) ◽

pp. 33-37 ◽

Cited By ~ 9

Author(s):

Y. Ii ◽

A. Uragami ◽

Y. Uno ◽

M. Kanechi ◽

N. Inagaki

Keyword(s):

Molecular Markers ◽

Sex Determination ◽

Genomic Dna ◽

Asparagus Officinalis ◽

Breeding Line ◽

Male And Female ◽

Future Studies ◽

Random Primers

Asparagus (Asparagus officinalis L.) plants are dioecious. All-male cultivars are desired because of their higher yields. To increase the proportion of male individuals planted in the field and expedite the breeding of all-male cultivars in asparagus, development of generally applicable molecular markers to distinguish male and female individuals is required. Bulked genomic DNA samples from ten male (XY) and ten female (XX) plants was screened with 10-bp random primers. Of the 188 primers tested, the primer T35R54 produced a 1600-bp fragment observed only in male individuals. The specificity of this T35R54-1600 marker was verified using DNA from one supermale (YY) and one female (XX) breeding line and their four F<sub>1</sub> progenies (XY). The T35R54-1600 marker fragment was observed in both supermale and all-male lines. The sequence of the T35R54 primer (5'-TTCACGGTGG-3') was absent among the sequences of primers or amplified fragments from previous studies. Therefore, this marker could be useful as a sex-related marker in future studies to increase the reliability of sex determination in asparagus.

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