scholarly journals Molecular Characterization of Mycoplasma arthritidis Variable Surface Protein MAA2

1998 ◽  
Vol 66 (6) ◽  
pp. 2576-2586 ◽  
Author(s):  
Leigh Rice Washburn ◽  
Keith E. Weaver ◽  
Elizabeth J. Weaver ◽  
Wendy Donelan ◽  
Suhaila Al-Sheboul
Keyword(s):  
Amino Acid ◽  
Signal Peptide ◽  
Dna Sequences ◽  
Surface Protein ◽  
Carboxypeptidase Y ◽  
Coding Region ◽  
Intact Cells ◽  
Mycoplasma Arthritidis ◽  
E Coli ◽  
Variable Surface

Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed inEscherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidisclonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative −10 box. Full-sized recombinant MAA2 was expressed inE. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

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

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 139-149 ◽  
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.

scholarly journals Genetic polymorphism of the extracellular region in surface associated interspersed 1.1 gene of Plasmodium falciparum field isolates from Thailand

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Natpasit Chaianantakul ◽  
Tippawan Sungkapong ◽  
Jirapinya Changpad ◽  
Keawalin Thongma ◽  
Sasiwimon Sim-ut ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequences ◽  
Balancing Selection ◽  
Haplotype Diversity ◽  
Research Area ◽  
Terminal Region ◽  
Common Amino Acid ◽  
Rich Domain ◽  
Extracellular Region ◽  
Variable Surface

Abstract Background A novel variable surface antigens (VSAs), Surface-associated interspersed proteins (SUFRINs), is a protein that is modified on the surface of infected red blood cell (iRBC). Modified proteins on the iRBC surface cause severe malaria, which can lead to death throughout the life cycle of a malaria parasite. Previous study suggested that SURFIN1.1 is an immunogenic membrane-associated protein which was encoded by using the surf1.1 gene expressed during the trophozoite and schizont stages. This study aimed to identify the regions of SURFIN1.1 and investigate the genetic diversity of the extracellular region of the surf1.1 gene. Methods A total of 32 blood samples from falciparum malaria cases that were diagnosed in Si Sa Ket Province, Thailand were collected. Plasmodium genomic DNA was extracted, and the extracellular region of surf1.1 gene was amplified using the polymerase chain reaction (PCR). A sequence analysis was then performed to obtain the number of haplotypes (H), the haplotype diversity (Hd), and the segregating sites (S), while the average number of nucleotide differences between two sequences (Pi); in addition, neutrality testing, Tajima’s D test, Fu and Li’s D* and F* statistics was also performed. Results From a total of 32 patient-isolated samples, 31 DNA sequences were obtained and analysed for surf1.1 gene extracellular region polymorphism. Researchers observed six distinct haplotypes in the current research area. Haplotype frequencies were 61.3%, 16.2%, and 12.9% for H1, H2, and H3, respectively. The remaining haplotype (H4-H6) frequency was 3.2% for each haplotype. Hd was 0.598 ± 0.089 with the Pi of 0.00381, and S was 15. The most common amino acid polymorphic site was E251Q; other sites included N48D, I49V, E228D, E235S, L265F, K267T, E276Q, and S288F. Fu and Li’s D* test value was − 1.24255, Fu and Li’s F* test value was − 1.10175, indicating a tendency toward negative balancing selection acting on the surf1.1 N-terminal region. The most polymorphic region was variable 2 (Var2) while cysteine-rich domain (CRD) was conserved in both the amino acid and nucleotide extracellular region of surf1.1 gene. Conclusions The Thai surf1.1 N-terminal region was well-conserved with only a few polymorphic sites remaining. In this study, the data regarding current bearing on the polymorphism of extracellular region of surf1.1 gene were reported, which might impact the biological roles of P. falciparum. In addition, may possibly serve as a suitable candidate for future development of SURFIN-based vaccines regarding malaria control. Graphic abstract

Partial cDNA sequence for the donkey chorionic gonadotrophin-β subunit suggests evolution from an ancestral LH-β gene

1990 ◽  
Vol 4 (2) ◽  
pp. 143-150 ◽  
Author(s):  
S. E. A. Leigh ◽  
F. Stewart
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Dna Sequences ◽  
Biological Activities ◽  
Chorionic Gonadotrophin ◽  
Amino Acid Residues ◽  
Β Subunit ◽  
Coding Region ◽  
Amino Acid Homology ◽  
Nucleotide Insertions

ABSTRACT A 246 bp cDNA clone representing the C-terminal region of the donkey (Equus asinus) chorionic gonadotrophin (CG)-β subunit was isolated from a placental library. The transcript contained the 3′ untranslated region and 42% of the CG-β subunit coding region (amino acid residues 85–146 of the mature peptide). Comparison of the deduced donkey amino acid sequence with the published horse CG-β subunit protein sequence (where they overlapped) revealed an overall homology of 61%. However, most of the differences were in the C-terminal extension, which is thought not to be important for gonadotrophic activity, and appeared to be due to two nucleotide insertions in the donkey sequence (compared with a deduced horse nucleotide sequence) leading to a reading-frame shift. Amino acid homology in the disulphide 'core' region was 81%. Some of the differences in this region were in the 'determinant loop' (residues 93–100) and these are interpreted in relation to the observed biological activities of horse and donkey CG. The deduced amino acid sequence of the donkey cDNA indicated that it was larger than the majority of gonadotrophin-β subunits due to a C-terminal extension. Primate and horse CG (and horse LH) β subunits have analogous C-terminal extensions. The extension in the donkey subunit is 25 amino acid residues in length, compared with 28 in the horse and 24 in man. Comparisons with other available subunit DNA sequences indicated that, like the human CG-β gene, the donkey gene probably evolved from an ancestral LH-like β gene, following nucleotide deletions that allowed readthrough into previously untranslated DNA. Furthermore, both the human and donkey CG-β genes make use of the original LH polyadenylation sequence AAUAAA for translational termination and polyadenylation. We conclude that the C-terminal extension arose independently in equids and primates but through similar mechanisms.

scholarly journals Identification and Characterization of Novel Surface Proteins in Lactobacillus johnsonii and Lactobacillus gasseri

2002 ◽  
Vol 68 (12) ◽  
pp. 6172-6181 ◽  
Author(s):  
Marco Ventura ◽  
Ivana Jankovic ◽  
D. Carey Walker ◽  
R. David Pridmore ◽  
Ralf Zink
Keyword(s):  
Amino Acid ◽  
Blot Analysis ◽  
Surface Protein ◽  
Promoter Sequence ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Lactobacillus Johnsonii ◽  
Intact Cells ◽  
Lactobacillus Gasseri ◽  
Cell Extracts

ABSTRACT We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.

Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins.

Genetics ◽  
1992 ◽  
Vol 132 (4) ◽  
pp. 963-973 ◽  
Author(s):  
R A Reenan ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Mismatch Repair ◽  
Dna Sequences ◽  
Amino Acid Sequences ◽  
Binding Motif ◽  
E Coli ◽  
Conserved Regions ◽  
Isolation And Characterization ◽  
Degenerate Oligonucleotide

Abstract Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms. Degenerate oligonucleotide primers based on conserved regions of E. coli MutS protein and its homologs from Salmonella typhimurium, S. pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae. Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS. These sequences were then used to clone the full-length genes from a yeast genomic library. Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp. The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively. The overall amino acid sequence identity with the E. coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2. Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs. Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair.

scholarly journals Characterization of human interleukin 2 derived from Escherichia coli

1985 ◽  
Vol 229 (2) ◽  
pp. 429-439 ◽  
Author(s):  
S M Liang ◽  
B Allet ◽  
K Rose ◽  
M Hirschi ◽  
C M Liang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Sequence ◽  
Protein A ◽  
Interleukin 2 ◽  
Coding Region ◽  
Disulphide Bridge ◽  
E Coli ◽  
Escherichia Coli Cells

Interleukin 2 isolated from Escherichia coli cells expressing the human interleukin gene has been characterized. The observed properties of the protein have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural human interleukin 2. The purified E. coli-derived interleukin 2 is a monomeric protein of Mr 15 000 with a sedimentation velocity of 1.86S. The amino acid composition of the protein and isoelectric point (7.7) are consistent with that part of the translated DNA sequence of the gene corresponding to the mature protein. A single disulphide bridge was identified between Cys-58 and Cys-105. C.d. suggested that interleukin 2 is predominantly alpha-helical in secondary structure. The E. coli-derived protein differed from natural interleukin 2 in the presence of N-terminal methionine and also in the absence of a carbohydrate moiety. Removal of the coding region for the first three amino acids of the natural interleukin 2 protein sequence (Ala-Pro-Thr) by site-specific mutagenesis resulted in a protein with N-terminal serine. The possibility that the specificity of the E. coli ribosomal methionine aminopeptidase may not recognize the sequence NH2-Met-Xaa-Pro is discussed (where Xaa is any amino acid residue).

scholarly journals Antigenic and Molecular Conservation of the Gonococcal NspA Protein

1999 ◽  
Vol 67 (6) ◽  
pp. 2855-2861 ◽  
Author(s):  
Martin Plante ◽  
Nathalie Cadieux ◽  
Clément R. Rioux ◽  
Josée Hamel ◽  
Bernard R. Brodeur ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Protein A ◽  
Surface Protein ◽  
Meningococcal Infection ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Intact Cells ◽  
Reading Frame ◽  
Calculated Molecular Weight

ABSTRACT A low-molecular-weight protein named NspA (neisserial surface protein A) was recently identified in the outer membrane of allNeisseria meningitidis strains tested. Antibodies directed against this protein were shown to protect mice against an experimental meningococcal infection. Hybridization experiments clearly demonstrated that the nspA gene was also present in the genomes of the 15 Neisseria gonorrhoeae strains tested. Cloning and sequencing of the nspA gene of N. gonorrhoeaeB2 revealed an open reading frame of 525 nucleotides coding for a polypeptide of 174 amino acid residues, with a calculated molecular weight of 18,316 and a pI of 10.21. Comparison of the predicted amino acid sequence of the NspA polypeptides from the gonococcal strains B2 and FA1090, together with that of the meningococcal strain 608B, revealed an identity of 93%, suggesting that the NspA protein is highly conserved among pathogenic Neisseria strains. The level of identity rose to 98% when only the two gonococcal predicted NspA polypeptides were compared. To evaluate the level of antigenic conservation of the gonococcal NspA protein, monoclonal antibodies (MAbs) were generated. Four of the seven NspA-specific MAbs described in this report recognized their corresponding epitope in 100% of the 51 N. gonorrhoeae strains tested. Radioimmunobinding assays clearly indicated that the gonococcal NspA protein is exposed at the surface of intact cells.

uspA of Shigella sonnei

2007 ◽  
Vol 70 (10) ◽  
pp. 2392-2395 ◽  
Author(s):  
JINRU CHEN
Keyword(s):  
Heavy Metals ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Stress Protein ◽  
Shigella Sonnei ◽  
Single Amino Acid ◽  
Coding Region ◽  
E Coli ◽  
Stress Responding ◽  
K 12

One of the strategies that bacteria utilize to combat environmental stress is to synthesize stress-responding proteins. In Escherichia coli, adverse environmental factors, such as starvation, heat, and the presence of acid, oxidants, heavy metals, and antibiotics, trigger the expression of the universal stress protein (USP). The gene of the USP, uspA, in E. coli K-12 and E. coli O157:H7 has been identified and sequenced. In this study, the nucleotide sequence of uspA in a strain of Shigella sonnei implicated in the 1998 parsley-related outbreak of shigellosis was determined. Within an 800-bp region sequenced, there were 17 bp mismatches between the uspA of S. sonnei and that of E. coli K-12. Among the 17 mismatched nucleotides, 8 were within the structure gene of uspA. A total of 12 bp variations were identified between the uspA of S. sonnei and that of E. coli O157:H7, of which 5 bp were internal to the coding region of uspA. However, unlike the mismatches between the uspA of E. coli K-12 and the same gene of E. coli O157:H7 and S. sonnei that resulted in a single amino acid substitution and changed an alanine to an arginine at position 140, the mismatches between S. sonnei and E. coli O157:H7 were silent and did not result in any amino acid substitution.

scholarly journals Characterization of Lactobacillus coryniformis DSM 20001T Surface Protein Cpf Mediating Coaggregation with and Aggregation among Pathogens

2004 ◽  
Vol 70 (12) ◽  
pp. 7078-7085 ◽  
Author(s):  
Martina Schachtsiek ◽  
Walter P. Hammes ◽  
Christian Hertel
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Surface Protein ◽  
Proteinase K ◽  
Northern Analysis ◽  
Phenotypic Characterization ◽  
Degenerate Primers ◽  
E Coli ◽  
Lactobacillus Coryniformis

ABSTRACT Phenotypic characterization of aggregation phenotypes of Lactobacillus coryniformis revealed that strain DSM 20001T coaggregated with Escherichia coli K88, Campylobacter coli, and Campylobacter jejuni but not with other human pathogens. In addition, cells of these pathogens aggregated in the presence of the spent culture supernatant (SCS) of strain DSM 20001T. Cells of E. coli K88 remained viable in the coaggregates and aggregates for up to 24 h. Both coaggregation and aggregation (co/aggregation) occurred at pH 3.5 to 7.5 and was sensitive to heat (85°C for 15 min) and proteinase K. The co/aggregation-promoting factor (Cpf) was purified, and the gene was identified by PCR with degenerate primers derived from internal amino acid sequences. The cpf gene encoded a 19.9-kDa preprotein with a sec-dependent leader and an isoelectric point of 4.4. The amino acid sequence had no significant similarity to proteins with known functions. Northern analysis revealed not only major transcription from the promoter of cpf but also major transcription from the promoter of the preceding insertion element, ISLco1 belonging to the IS3 family. Recombinant Cpf produced in E. coli mediated aggregation of pathogens comparable to the aggregation obtained with purified Cpf or SCS of strain DSM 20001T. Cpf could be removed from cells of strain DSM 20001T by treatment with 5 M LiCl and could be subsequently reattached to the cell surface by using SCS or recombinant Cpf, which resulted in restoration of the co/aggregation property. These results together with those of the amino acid sequence analysis suggest that Cpf is a novel surface protein of L. coryniformis that mediates co/aggregation of some pathogens.

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