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PubMed1999

Sequencing of HLA class II genes based on the conserved diversity of the non-coding regions: sequencing based typing of HLA-DRB genes.

In this paper, we present a novel sequencing based typing strategy for the HLA-DRB1, 3, 4 and 5 loci. The new approach is based on a group-specific amplification from intron 1 to intron 2 according to the serologically-defined antigens. For this purpose, we have determined the 3' 500 bp-fragment of intron 1 and the 5' 340 bp-fragment of intron 2 of all serological antigens and their most frequent subtypes. We discovered a remarkably conserved diversity characterized by lineage-specific sequence motifs. This lineage-specificity of non-coding motifs in the 1st and 2nd intron offered the possibility to establish a clear serology-related amplification strategy. The method allows the complete analysis of the 2nd exon and the definition of the cis/trans linkage of sequence motifs by intron-mediated polymerase chain reaction (PCR)-based separation of the haplotypes in nearly all serologically heterozygous samples. In particular, the non-coding variabilities between the DR52-associated DRB1 groups made their independent amplification possible. Thus, compared to the standard procedures using exon-based amplification primers, the groups DR3, DR12, some DR13 alleles (1301, 1302) and the DR14 group could be amplified by specific primer mixes. The DR8 could be amplified with an individual primer mix not co-amplifying the DR12. The DR11 and DR13 did not show any individual motif in intron 1 or intron 2. In order to achieve a separate amplification, they had to be amplified by multispecific primer mixes (DR3/11/13/14; DR3/11/13 or DR11/13/14) excluding the other haplotype. Thus, exclusively the alleles in rare DR11,13 heterozygosities without a DRB1*1301 or 1302 could not be amplified separately. Fourteen primer mixes are used to amplify the specificities DR1-14, and 6 primer mixes for the specificities DR51-53. The sequence homology of the 3' end of intron 1 facilitated the application of only three different sequencing primers for all DRB alleles.

Alleles↗
PubMed2003

Sequence-based typing identifies a novel HLA-DPB1 allele, DPB1*9601.

In this report we describe the identification of a novel HLA-DPB1 allele, DPB1*9601, found in a Caucasian individual sample named ucla#356. The new allele was detected in the DNA of ucla#356 during routine HLA sequence-based typing (SBT) of samples participating in the UCLA International HLA DNA Exchange (number 55) for HLA DNA Proficiency Testing. DPB1*9601 was identical to DPB1*3901 except for a single nucleotide substitution 'G'-->'C' in previously constant position 277 (position 177, respectively, counting only exon 2). This nucleotide change causes an amino acid substitution from aspartic acid in DPB1*3901 to histidine at codon 64 in the novel allele. This new allele has been submitted to the EMBL database and has been assigned the accession number AJ514871. The WHO Nomenclature Committee has officially assigned the name DPB1*9601.

Base Sequence↗
PubMed2002

Distribution of fim3 and flaA TTGE sequence types amongst isolates of Bordetella bronchiseptica from different host animals.

Isolates of Bordetella bronchiseptica associated with different animal hosts were analysed by fim3 and flaA genotyping by temporal temperature gradient gel electrophoresis (TTGE). All the isolates from cats (n = 138), dogs (n = 42) and pigs (n = 13) could be assigned to one of two fim3 and one of three flaA TTGE sequence types, respectively. Two avian isolates and a marmoset isolate exhibited novel fim3 sequence types. Dominant but different TTGE sequence types were apparent in isolates from dogs and pigs for both fim3 (87.5% and 100%, respectively, of isolates were the dominant type) and flaA (95% and 92%, respectively, of isolates were the dominant type). There was a wider distribution of TTGE sequence types amongst cat isolates. As both fimbriae and flagella have been implicated in adherence of bordetellae to host cells, sequence variations in fimbrial proteins and FlaA may have a role to play in host preferences.

Amino Acid Sequence↗
PubMed1998

Sequencing-based typing reveals six novel MHC class I chain-related gene B (MICB) alleles.

The MHC class I chain-related gene A (MICA) is highly polymorphic. In this paper we demonstrate polymorphism in the other expressing member of the MIC family of genes: MICB. Using a sequencing-based typing approach on cDNA, analysis of exons 2 through 6 revealed eight polymorphic sites resulting in six unique MICB sequences. Although MICB has high nucleotide homology with MICA, its polymorphism is restricted and at different sites compared to those in MICA.

Alleles↗
PubMed2006

Sequence-based typing reveals a novel DLA-88 allele, DLA-88*04501, in a beagle family.

The dog is an important animal model for solid organ as well as stem cell allo transplantation. Methods such as cellular and serological typing and more recently sequence-based typing (SBT) have been used to discriminate tissue antigen disparity of donor and recipient. We applied SBT for the canine class I (DLA-88) and class II (DLA-DRB1) genes in beagle families prior stem cell transplantation. A novel DLA-88 (DLA-88*04501) allele in combination with a DLA-DRB1*01901 allele was found. Sequence comparison of exons 2 and 3 of the novel allele revealed most sequence identity to the DLA-88*01301 allele (96.15% identity at the nucleotide and 90.65% identity at the protein level).

Alleles↗
PubMed2000

Three novel polymorphic sequence variants in the type I collagen gene COL1A1, the main disease locus for Osteogenesis Imperfecta.

Three novel polymorphic variants were found within COL1A1 genomic sequence (accession number AF017178) while screening several patients in the search of OI causal mutations. The three polymorphisms, located in intron 12, exon 26, and intron 29, respectively, can be detected by PCR amplification and digestion with appropriate restriction enzymes (Mbo II, Bst NI, Pvu II, respectively). Allelic frequencies within the Italian population were calculated.

Base Sequence↗
PubMed2006

Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs.

The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a "splash"-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohn's disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohn's disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile.

Animals↗
PubMed2025

Genomic insights into Shigella species isolated from small ruminants and manure in the North West Province, South Africa.

This study investigated Shigella species' antibiotic resistance patterns and genomic characteristics from small ruminants and manure collected in Potchefstroom, North West, South Africa. Whole genome sequencing was used to determine resistome profiles of Shigella flexneri isolates from small ruminants' manure and Shigella boydii from sheep faeces. Comparative genomics was employed on the South African 261 S. flexneri strains available from GenBank, including the sequenced strains in this study, by investigating the serovars, antibiotic resistance genes (ARGs), and plasmid replicon types. The S. flexneri strains could not be assigned to known sequence types, suggesting novel or uncharacterized lineages. S. boydii R7-1A was assigned to sequence type 202 (ST202). Serovar 2A was the most common among South African S. flexneri strains, found in 96% of the 250 compared human-derived isolates. The shared mdf(A) was the most prevalent gene, identified in 99% of 261 S. flexneri genomes, including plasmid replicon types ColRNAI_1 (99%) and IncFII_1 (98%). Both species share a core set of resistance determinants mainly involving β-lactams (ampC1, ampC, ampH), macrolides (mphB), polymyxins (eptA, pmrF), multidrug efflux pumps (AcrAB-TolC, Mdt, Emr, Kpn families), and regulatory systems (marA, hns, crp, baeRS, evgAS, cpxA, gadX). However, S. boydii possesses additional resistance genes conferring resistance to tetracyclines (tet(A)), phenicols (floR), sulphonamides (sul2), and aminoglycosides (APH(3'')-Ib, APH(6)-Id), along with the acrEF efflux pump components (acrE, acrF). In contrast, S. flexneri harboured unique genes linked to polymyxin resistance (ugd) and regulatory functions (sdiA, gadW) that were absent in S. boydii. These findings highlight Shigella strains' genomic diversity and antimicrobial resistance potential in livestock-associated environments. Moreover, S. boydii highlights the potential risk of multidrug-resistant bacteria in farming and environmental routes. KEY POINTS: • First whole genome study of Shigella from manure and small ruminants in South Africa. • Shigella boydii strain carried multiple resistance genes to β-lactams and tetracycline. • Multidrug efflux pump gene mdf(A) was detected in 99% of South African Shigella flexneri strains.

Animals↗
PubMed1992

Polymerase chain reaction based mapping of rye involving repeated DNA sequences.

A novel type of polymerase chain reaction (PCR) marker was developed for the mapping of cereal rye (Secale cereale). Primer pairs were synthesized targeting the insertion sites of three individual copies of the R173 family of rye specific repeated DNA sequences. While one primer was derived from a sequence within the respective R173 element, the second primer corresponded to a flanking region. The complex banding patterns obtained in rye allowed not only the mapping of the three R173 elements to certain chromosome regions of 1RS (the short arm of rye chromosome 1) but also the mapping of an additional 3-10 easily identifiable bands per primer pair to other rye chromosomes. Linkage mapping of a polymorphic 1R band derived from three rye cultivars demonstrated the presence of nonallelic, dominant markers in two independent crosses. Because of the high copy number of the R173 family (15,000 copies per diploid rye genome), its dispersion over the entire length of all chromosomes and the high number of markers obtained per primer pair, PCR markers based on the R173 family provide an almost unlimited source for well-spaced markers in rye mapping.

Base Sequence↗
PubMed2001

Identification of a novel DRB1 allele, DRB1*1112, by sequence-based DRB typing.

We report here a novel DRB1 allele (DRB1*1112) identified during sequence-based HLA-DRB typing. Polymerase chain reaction with generic DRB primers and group-specific primers and subsequent sequencing yielded identical results. Molecular cloning and sequencing confirmed that the new DRB1 allele is identical to DRB1*11011 and 1129 at exon 2 except for a single nucleotide substitution at codon 37, changing the codon from Tyr (DRB1*11011) or Ser (DRB1*1129) to Phe.

Aged↗
PubMed2003

High resolution sequence-based DPB1 typing identified two novel DPB1 alleles, DPB1*9401 and DPB1*9501, from a Kenyan population.

Two novel DPB1 alleles, DPB1*9401 and DPB1*9501, were identified from a Kenyan population during sequence-based HLA-DPB1 typing. Molecular cloning and sequencing of multiple clones confirmed that one of the new DPB1 alleles is identical to DPB1*0402 at exon 2 except for a single nucleotide substitution (CGG -->TGG), changing codon 70 from Arg to Trp. The new allele has been named DPB1*9401. This is the first report of polymorphism at codon 70 of HLA-DPB1 alleles. New codon combinations have been identified in another novel DPB1 allele named DPB1*9501. The extensive diversity at DPB1 locus of this East African population is being revealed by high resolution sequence-based DPB1 typing.

Amino Acid Sequence↗
PubMed2005

High-resolution sequence-based DPA1 typing identified two novel DPA1 alleles, DPA1*010303 and DPA1*0303, from a Kenyan population.

We report here two novel DPA1 alleles, DPA1*010303 and DPA1*0303, identified from a Kenyan population during sequence-based HLA-DPA1 typing. Molecular cloning and sequencing of multiple clones confirmed that one of the new DPA1 alleles is identical to DPA1*010301 at exon 2, except for a single nucleotide substitution (ACG ACC) at codon 15. The new allele has been named by the WHO Nomenclature Committee as DPA1*010303. The second novel DPA1 allele is identical to DPA1*0301, except for a single nucleotide difference (GAA GAC) at codon 28 that changed the amino acid from Glu to Asp. The new allele has been named by the WHO Nomenclature Committee as DPA1*0303. Identification of the two novel DPA1 alleles reflects the genetic diversity of this East African population.

Base Sequence↗
PubMed2006

Clonal structure of Enterococcus faecalis isolated from Polish hospitals: characterization of epidemic clones.

To study the population structure of Enterococcus faecalis from Polish hospitals, 291 isolates were typed by pulsed-field gel electrophoresis and a novel multilocus sequence typing scheme (P. Ruiz-Garbajosa et al., J. Clin. Microbiol. 44:2220-2228, 2006). The isolates originated from geographically widespread medical institutions and were recovered during a 10-year period (1996 to 2005) from different clinical sources. The analysis grouped the isolates into five epidemic and 71 sporadic clones. The importance of the previously identified global clonal complexes CC2 and CC9 was corroborated by our findings that two of the Polish epidemic clones, A and J, were classified into these clonal complexes (CCs). However, the two most predominant clones, C (ST40) and F (CC87), did not cluster in the aforementioned CCs and may represent novel epidemic CCs. These clones may have emerged in Central Europe. Clone F, carrying glycopeptide resistance determinants of VanA or VanB phenotypes, caused several outbreaks in hematology units and appeared to be the most prevalent clone in recent years in Poland. Antimicrobial susceptibility testing and additional tests for pathogenicity-related phenotypes (hemolysin and gelatinase production) and genes (asa1 and esp) were performed to further characterize these epidemic clones. Multidrug resistance, glycopeptide resistance, presence of asa1, and production of hemolysin appeared to be statistically significant features related to epidemicity. Production of gelatinase was significant for two of the epidemic clones, whereas presence of the esp gene was not specific for the epidemic clones.

Anti-Bacterial Agents↗
PubMed2006

Identification of four novel HLA-A alleles from an East African population by high-resolution sequence-based typing.

We report here four novel human leukocyte antigen (HLA)-A alleles identified among an East African population during sequence-based HLA-A typing. The novel alleles were confirmed by sequencing two separate polymerase chain reaction products and by molecular cloning and sequencing multiple clones. The new allele A*9202 is identical to A*0202 at exon 2 and exon 3 except for a single nucleotide difference at codon 43 (CGG-->CAG), resulting in a coding change from Arginine to Glutamine. The second new allele has a synonymous change at codon 139 (GCA-->GCG), that differentiates it from A*680101. The new allele has been named by the World Health Organization nomenclature committee as A*680105. The novel allele A*2630 is identical to A*2603 at exon 2 and exon 3 except for a nonsynonymous change at codon 90 (GAC-->GCC), changed from Aspartic acid to Alanine. The fourth new allele is identical to A*290201 except for a single nucleotide difference at codon 138 (ATG-->GTG), resulting in a coding change from Methionine to Valine. The new allele has been named by the World Health Organization nomenclature committee as A*2915. Identification of these novel HLA-A alleles reflects the genetic diversity of this East African population.

Africa, Eastern↗
PubMed1999

Identification of a novel HLA-DQA1 allele (DQA1*0106) by sequence-based DQA1 typing.

We report here a novel DQA1 allele (DQA1*0106) identified during sequence-based HLA-DQA1 typing. Polymerase chain reaction with proofreading pfu DNA polymerase and subsequent sequencing yielded identical results as that with Taq DNA polymerase. Molecular cloning and sequencing confirmed that the new DQA1 allele is identical to DQA1*01021/2 at exon 2 except for a single nucleotide substitution (ACT-->GCT), changing codon 44 from Thr to Ala. This is the first report of polymorphism at codon 44 of HLA-DQA1 alleles.

Alanine↗