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PubMed2025

Allele Level Sequencing of Killer Cell Immunoglobulin-Like Receptor Genes Using Oxford Nanopore Long Read Sequencing.

The human Killer cell Immunoglobulin-like Receptor (KIR) genes, found on chromosome 19, encode for cell surface protein receptors that, through interaction with their ligand, modulate the action of Natural Killer (NK) cells and some subsets of T lymphocytes. KIR genes exhibit extensive variation through variable gene content, copy number, and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant, and infectious disease progression. KIR gene content has been used in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. With the introduction of massively parallel clonal next generation sequencing and single molecule long read third generation sequencing, allele level determination of KIR genotypes has become feasible. We describe a method for amplicon-based long read sequencing on the Oxford Nanopore Technologies platform that provides largely unambiguous allele level typing of KIR genes. The method was validated using DNA extracted from 48 10th International Histocompatibility Workshop (IHWS) cell lines with previously published allele level KIR genotypes and 176 Western Australian samples previously tested for the presence or absence of KIR genes. Our long-read sequencing method was able to accurately determine KIR alleles with an overall concordance of 97%-99% with the published data. Importantly, phasing ambiguity caused by the inability to phase heterozygous base positions over long stretches of gene sequence was resolved in several samples. Thus, our long read PCR sequencing strategy can be used to determine KIR genotypes at allele resolution level.

Humans↗

PubMed2003

Diversity in coding tandem repeats in related Neisseria spp.

BACKGROUND: Tandem repeats contained within coding regions can mediate phase variation when the repeated units change the reading frame of the coding sequence in a copy number dependent manner. Coding tandem repeats are those which do not alter the reading frame with copy number, and the changes in copy number of these repeats may then potentially alter the function or antigenicity of the protein encoded. Three complete neisserial genomes were analyzed and compared to identify coding tandem repeats where the number of copies of the repeat will have some structural consequence for the protein. This is the first study to address coding tandem repeats that may affect protein structures using comparative genomics, combined with a population survey to investigate which show interstrain variability. RESULTS: A total of 28 genes were identified. Of these, 22 contain coding tandem repeats that vary in copy number between the three sequenced strains, three strain specific genes were included for investigation on the basis of having >90% identity between repeated units, and three genes with repeated elements of >250 bp were included although no length variations were seen in the genomes. Amplification, and sequencing of repeats showing altered copy number, of these 28 coding tandem repeat containing regions, from a set of largely unrelated strains, revealed further repeat length variation in several cases. CONCLUSION: Eighteen genes were identified which have variation in repeat copy number between strains of the same species, twelve of which show greater diversity in repeat copy number than is present in the sequenced genomes. In some cases, this may reflect a mechanism for the generation of antigenic variation, as previously described in other species. However, some of the genes identified encode proteins with cytoplasmic functions, including sugar metabolism, DNA repair, and protein production, in which repeat length variation may have other functions. Coding tandem repeats appear to represent a largely unexplored mechanism of generating diversity in the Neisseria spp.

Adhesins, Bacterial↗

PubMed2006

[Changes in copy number and deletion development of mitochondrial DNA in blood cells from breast cancer patients during radiochemotherapy].

Changes in mitochondrial DNA (mtDNA) copy number were compared versus the nuclear beta-globulin gene (internal standard), as well as occurrence of large mtDNA deletions in peripheral blood samples from 21 breast cancer patients following chemoradiotherapy. The study used polymerase chain reaction. Distinct variations were identified both in mtDNA copy number versus nuclear DNA and large mtDNA deletions occurrence in blood cells in response to genotoxic influence of chemoradiotherapy. Some mtDNA copies in breast cancer patients blood cells revealed large deletions whose frequency increased after chemoradiotherapy. Mitochondrial DNA lesions would induce synthesis of mtDNA copies in the course of chemoradiotherapy, which pointed to a compensatory reaction in peripheral blood cells as a consequence of a disturbed energy biogenesis.

Adult↗

PubMed2000

Comparative genomic hybridization reveals extensive variation among different MCF-7 cell stocks.

Comparative genomic hybridization (CGH) allows the detection of DNA sequence copy number changes on a genome-wide scale in a single hybridization reaction. The ability of CGH to be applied to formalin-fixed, paraffin-embedded tumor samples has lead to its widespread application in the cytogenetic analysis of archival material. When setting up CGH in the laboratory, rigorous control experiments must be carried out to ensure that the losses and gains are scored correctly. Groups interested in breast cancer frequently use the MCF-7 cell line as a positive control in these experiments, comparing the results to previously described genetic alterations. Here we present the results of CGH carried out with three stocks of MCF-7 cells. The cells differ widely in their proliferative response to 17-beta estradiol and show extensive variation in copy number changes affecting specific chromosomal regions. We suggest that care must be taken, therefore, when choosing a cell line as a positive control for CGH experiments.

Breast Neoplasms↗

PubMed2001

Extraordinary variation in a diversified family of immune-type receptor genes.

Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.

Amino Acid Sequence↗

PubMed1997

Variant of intron 22 inversions in the factor VIII gene in severe hemophilia A.

Recurrent DNA inversions, which disrupt the factor VIII (FVIII) gene, generally occur between a region of intron 22 (int22h) and one of two homologous copies of this region, located 300 to 400 kb telomeric to the FVIII gene. This report describes a patient with severe hemophilia A and a high level inhibitor with atypical hybridization patterns. A Bcl I Southern blot assay was altered to 17.5, 16, and 14 kb. His mother and two out of four aunts tested had normal and abnormal restriction patterns which led to a total of five different fragments, suggesting that they were carriers. The Xba I plus Kpn I restriction fragment-length polymorphism in intron 22 by Southern blotting using the same probe (probe a) yielded the 6.2 kb polymorphic band, with a clearly separated 6.6 kb band from the non-factor VIII region; an alternative int22h hybridization probe (probe x) detected no additional fragment. These results suggest that probe a as well as probe x could recognize an intron-22-sized fragment. This report shows a variation in the number of int22h copies although we could not find the inversion junction.

Adult↗

PubMed1990

Molecular characterization of a repeat element causing large-scale size variation in the mitochondrial DNA of the sea scallop Placopecten magellanicus.

The scallop Placopecten magellanicus has the largest reported animal mitochondrial DNA (average 35 kb) and exhibits large inter- and intraindividual length variation owing to the varying copy number of a repeated element. We have characterized the repeat array by using restriction mapping and sequence analysis. The repeated element consists of 1,442 bp flanked on either side by the sequence ACTTTCC in a direct orientation. The array contains two to eight copies of the repeated element arranged in a direct orientation and in tandem. Only complete copies of the element are present in the array. The repeat element contains three regions with characteristic nucleotide sequences: a 10-bp inverted repeat shown to extrude into a cruciform in a supercoiled DNA plasmid, a 120-bp tract rich in G/C (70%) and adjacent to the inverted repeat, and periodically interspersed homopolymer runs of A and T occurring near the middle of the element which induce DNA curvature in dimeric constructs of the element. The element appears to be unique to P. magellanicus. The structural properties of the repeat element and its organization in an array of repeats may be important in explaining the generation and maintenance of large-scale mitochondrial DNA size variation observed in many animal species.

Animals↗

PubMed1995

Variation in ribosomal DNA intergenic spacers among populations of Anopheles albimanus in South and Central America.

Variation in the length and copy number of intergenic spacers (IGS) of nuclear ribosomal DNA were examined to test for genetic differentiation among Anopheles albimanus populations. Extensive collections were made in Guatemala but populations were also sampled over a large range of its distribution in Central and South America. Discriminant analysis of IGS patterns in individual mosquitoes indicated that populations generally had unique sets of IGS length variants. The IGS patterns from populations on the Pacific side of Central America were distinct from those on the Atlantic side or from South America. Cluster analysis indicated a similar trend. The IGS diversity in Central America was 50% greater than in South America. These results suggest that barriers to gene flow exist among Atlantic and Pacific coast populations of An. albimanus. No gene flow barriers were detected among populations from Colombia, Ecuador, Peru, and Venezuela.

Animals↗

PubMed2005

Improved detection of grapevine leafroll-associated virus 1 by magnetic capture hybridisation RT-PCR on a conserved region of viral RNA.

We report the development of a sensitive diagnostic method for the detection of the grapevine leafroll-associated virus 1 (GLRaV-1). We have considered the current shortcoming in detection of GLRaV-1 to be linked to two factors, sequence variation in the viral RNA and low template concentration. Sequence information available allowed the selection of optimal target sequences for detection by RT-PCR, having high copy number and low levels of sequence variation. This was combined with the use of magnetic capture hybridisation to allow the removal of RT-PCR inhibitors and the addition of 100-fold excess template RNA to a single RT-PCR. The reproducibility of the technique was confirmed using field samples.

Closteroviridae↗

PubMed1990

Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus).

A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.

Animals↗

PubMed2000

Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris.

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. This enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. An improved method of production of recombinant alpha-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human alpha-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human alpha-galactosidase A. Recombinant human alpha-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25 degrees C, but not at the standard growth temperature of 30 degrees C. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant alpha-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.

Bioreactors↗

PubMed1990

Autonomous transposition of gypsy mobile elements and genetic instability in Drosophila melanogaster.

The laboratory mutator strain (MS) of Drosophila melanogaster is characterized by an elevated frequency of spontaneous mutation (10(-3)-10(-4). Mutations occur in both sexes at premeiotic stages of germ cell development. The increased mutability is a characteristic feature of MS itself, since it appears in the absence of outcrossing. Most of the mutations arising in this strain are unstable: reversions to wild type, high frequency mutation to new mutant states and replicating instability were observed. We have investigated the localization of the transposable genetic elements mdg1, 412, mdg3, gypsy (mdg4), copia and P in the X chromosomes of the MS and in the mutant lines y, ct, sbt derived from it by in situ hybridization. The P element was not found in any of these strains. The distributions of mdg1, 412, mdg3 and copia were identical in the X chromosomes of the MS and its derivatives. However, the sites of hybridization with gypsy differ in the various lines tested. In the polytene chromosomes of MS animals significant variation in location and number of copies of the gypsy element was demonstrated between different larvae; copy numbers as high as 30-40 were observed. These results suggest autonomous transposition of gypsy in the MS genome while several other mobile elements remain stable.

Animals↗

PubMed1985

Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant.

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.

Bacteriophages↗

PubMed1994

Transposon-induced rearrangements in the duplicated locus ph of Drosophila melanogaster can create new chimeric genes functionally identical to the wild type.

Variation in the number of gene copies can play a major role in changing the coding capacities of eukaryotic genomes. Different mechanisms, such as unequal recombination or transposon-induced chromosome rearrangements, are believed to be responsible for these events. We have used the direct tandem duplication at the complex locus polyhomeotic (ph) of Drosophila melanogaster as a model system to study functional redundancy associated with chromosomal rearrangements, such as duplications or deletions. The locus covers 28.6 kb and comprises two independent units, ph proximal and ph distal, which are not only similar on the molecular level, but appear to be functionally redundant [Dura et al., Cell 51 (1987) 829-839; Deatrick et al., Gene 105 (1991) 185-195]. We present a molecular and phenotypic analysis of two hypomorphic ph mutants, ph2 and ph4, induced during hybrid dysgenesis. Each corresponds to an internal deletion in the ph locus that overlaps both transcription units. We show that the deletions are likely due to a P/M hybrid dysgenesis-induced rearrangement between proximal and distal ph, that created a single new chimerical ph gene. At least one of the breakpoints must be located in a 1247-bp region that is rich in single sequence, and 100% identical between proximal and distal ph. Junction points between units are in the protein-coding regions, but could not be exactly localized on the genomic sequence of either mutant, because of the precise molecular mechanism that caused the deletions. Protein products of the hybrid genes contain the same functional domains as either wild-type (wt) product.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1999

Size-polymorphism of mini-exon gene-bearing chromosomes among natural populations of Leishmania, subgenus Viannia.

In order to explore genomic plasticity at the level of the mini-exon gene-bearing chromosome in natural populations of Leishmania, the molecular karyotype of 84 Leishmania stocks belonging to subgenus Viannia, originating mostly from Peru and Bolivia, and differing according to eco-geographical and clinical parameters, was resolved and hybridised with a mini-exon probe. The results suggest that size variation of the mini-exon gene-bearing chromosome is frequent and important (up to 245-kb size-difference), and partially involves variation (up to 50%) in copy number of mini-exon genes. There is no significant size-difference between mini-exon-bearing chromosomes of Peruvian and Bolivian populations of cutaneous and mucosal isolates of Leishmania (Viannia) braziliensis, but there is between eco-geographical populations of Leishmania (Viannia) peruviana. Leishmania (V.) peruviana presented a significantly smaller mini-exon-bearing chromosome than the other species of subgenus Viannia. The contrast between the general chromosome size heterogeneity and the homogeneity observed in some Peruvian Andean areas is discussed in terms of selective pressure.

Animals↗

PubMed2001

Network analysis provides insights into evolution of 5S rDNA arrays in Triticum and Aegilops.

We have used network analysis to study gene sequences of the Triticum and Aegilops 5S rDNA arrays, as well as the spacers of the 5S-DNA-A1 and 5S-DNA-2 loci. Network analysis describes relationships between 5S rDNA sequences in a more realistic fashion than conventional tree building because it makes fewer assumptions about the direction of evolution, the extent of sexual isolation, and the pattern of ancestry and descent. The networks show that the 5S rDNA sequences of Triticum and Aegilops species are related in a reticulate manner around principal nodal sequences. The spacer networks have multiple principal nodes of considerable antiquity but the gene network has just one principal node, corresponding to the correct gene sequence. The networks enable orthologous groups of spacer sequences to be identified. When orthologs are compared it is seen that the patterns of intra- and interspecific diversity are similar for both genes and spacers. We propose that 5S rDNA arrays combine sequence conservation with a large store of mutant variations, the number of correct gene copies within an array being the result of neutral processes that act on gene and spacer regions together.

Algorithms↗

PubMed2004

NUMTs in sequenced eukaryotic genomes.

Mitochondrial DNA sequences are frequently transferred to the nucleus giving rise to the so-called nuclear mitochondrial DNA (NUMT). Analysis of 13 eukaryotic species with sequenced mitochondrial and nuclear genomes reveals a large interspecific variation of NUMT number and size. Copy number ranges from none or few copies in Anopheles, Caenorhabditis, Plasmodium, Drosophila, and Fugu to more than 500 in human, rice, and Arabidopsis. The average size is between 62 (baker's yeast) and 647 bps (Neurospora), respectively. A correlation between the abundance of NUMTs and the size of the nuclear or the mitochondrial genomes, or of the nuclear gene density, is not evident. Other factors, such as the number and/or stability of mitochondria in the germline, or species-specific mechanisms controlling accumulation/loss of nuclear DNA, might be responsible for the interspecific diversity in NUMT accumulation.

Cell Nucleus↗

PubMed2004

Quantitative real-time polymerase chain reaction based on single copy gene sequence for detection of periodontal pathogens.

OBJECTIVE: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. MATERIAL AND METHODS: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (C(T)) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. RESULTS: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (C(T)) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. CONCLUSION: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samples.

Aggregatibacter actinomycetemcomitans↗