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PubMed2000

PCR-mediated gene replacement in Escherichia coli.

The hyper-recombinogenic properties of an E. coli strain in which the recBCD genes have been replaced by lambda red recombination functions were exploited in the development of a general PCR-mediated gene replacement scheme for Escherichia coli. Linear DNA substrates generated by recombinant PCR are introduced by electroporation into strains containing the recBCDDelta::red substitution. This technique allows for gene replacement in E. coli without prior cloning of the gene of interest. In addition, the counter-selectable marker sacB has been used to construct unmarked precise gene deletions without the need to form sacB-containing plasmid integrates. In other experiments, electroporation of recBCDDelta::red strains with high concentrations of linear DNA fragments (derived from plasmid digests) gave linear transformation rates approaching 1% of the survivors of electroporation. The placement of lambda red and gam at a locus in the chromosome other than recBCD (galK) resulted in a strain that was as hyper-rec as one containing the lambda red for recBCD substitution. The gene replacement technique described here has been used for the construction of deletion-substitution alleles of lacZ and sulA, as well as six genes important for general homologous recombination in E. coli. Three of these replacements were performed without prior cloning of the genes.

Bacteriophage lambda↗
PubMed2006

Routine markerless gene replacement in Bacillus anthracis.

An improved genetic tool suitable for routine markerless allelic exchange in Bacillus anthracis has been constructed. Its utility was demonstrated by the introduction of insertions, deletions, and missense mutations on the chromosome and plasmid pXO1 of the Sterne strain of B. anthracis.

Bacillus anthracis↗
PubMed2005

A novel A*24 allele, A*2442, was detected through routine bone marrow donor screening.

In this article, we report the identification of a new HLA-A allele found in a DNA sample which was part of the routine bone marrow donor typing performed in our laboratory. This novel allele officially designated as A*2442 was found in a sample from a female Caucasoid donor (Franken, Bavaria, Germany; lab code 142654) and differs from the closest related allele A*2408 by two nucleotide exchanges. In position 81, the A (A*2408) is changed to 81 C in the novel allele A*2442, resulting in an amino acid substitution at codon 27, glutamine (A*2408) is replaced by histidine ((31)Gln-->(31)His). In position 292, the G (A*2408) is changed to C also resulting in an amino acid replacement, the codon 98 asparagine is mutated to histidine ((98)Asn-->(98)His) in the new A*2442 allele. The second allele was determined to be A*0301. Further typing of this sample is B*1501 B*4001.

Alleles↗
PubMed1996

Host-parasite arms race in mutation modifications: indefinite escalation despite a heavy load?

If constantly changing genotypes are favorable in host and parasite coevolution, an indefinite escalation of mutation rates would result despite heavy mutational loads. We theoretically study this possibility by examining the mutation modifier dynamics of host and parasite that engage in genotype-specific epidemiological interaction. In the first model, we study the evolutionarily stable (ESS) mutation rate or switching rate if two alleles in a single locus are subjected to frequency-dependent selection favoring the rarer of the two. Mutation modifier locus is either tightly linked or unlinked to the selected locus. Sufficiently strong frequency-dependent selection may cause cycles in allele frequencies and a modifier with higher mutation rate enjoys the long-term advantage by randomizing the genotype of their offspring. Through the repeated events of invasion and replacement of mutation modifiers, the mutation rate continues to increase until the allele frequencies are stabilized. If some fraction of mutations are deleterious, there is no longer a pure ESS mutation rate: the evolutionarily stable population then consists of multiple strains concerning mutation modifier, typically one with a very high mutation rate and the other with a very low rate, stably coexisting and fighting off invasion by any other modifiers. These results are almost independent of the linkage between the selected and the modifier loci. In the second model, we consider the joint evolution of host and parasite mutation modifiers, assuming that a specific pair of host and parasite genotype densities change following the Nicholson-Bailey type model. If there is no cost of deleterious mutations, mutation rates of both species are escalated indefinitely by modifier evolution until they completely suppress the fluctuation of genotype densities. However, a small cost of deleterious mutation is enough to collapse this coevolutionary equilibrium of inflated mutations. Typical coevolutionary outcome is that the parasite mutation rate is accelerated to a high level; whereas the host mutation rate is driven to zero. Extension of our results to host-parasite coevolution of recombination modifier evolution is discussed.

Animals↗
PubMed1996

Epigenetic control of a transposon-inactivated gene in Neurospora is dependent on DNA methylation.

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5' noncoding sequences was found in previous work. We report that reversion to Am+ depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am+ conidia, whether the cultures were derived from Am- or Am+ isolates. Reversion to Am+ did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am+. The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSam alpha and beta, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.

Alleles↗
PubMed1999

Evolution of HLA-class I compared to HLA-class II polymorphism in Terena, a South-American Indian tribe.

We have studied the HLA alleles of 60 unrelated healthy Terena and 10 Terena families. They are members of an isolated Brazilian tribe located in Mato Grosso do Sul (South Central Brazil). Six novel alleles were found in this population: HLA-A*0219 (gf = 0.02), A*0222 (gf = 0.15), HLA-B* 3520 (gf = 0.01), B*3521 (gf = 0.03), B*3912 (gf = 0.03) and B*4803 (gf = 0.16). Five of the six novel alleles differ from their putative progenitors by amino acid replacements in residues that contribute to the pockets of the peptide-binding site. Many of the variants defined by molecular methods were not identified correctly by serological typing. We calculated heterozygosity values (H) for HLA-A, -B, -C, DRB1, DQB1 and DPB . The highest values were observed at the HLA-B locus, followed by HLA-A, -DRB1 and DQB1. Residue positions 9, 24, 45, 62, 67, 95, 114, 116, 156, and 163 of HLA class I showed heterozygosity values greater than 0.50. Nine of them contribute to the peptide-binding specificity pockets and one to the T cell receptor binding site. If HLA antigens are useful for defense against pathogenic agents, heterozygosity would offer an advantage by allowing binding of a larger repertoire of peptides to the class I molecules. Individuals that are heterozygous at these positions would probably have a wider repertoire of peptide presentation to T cells. The observed results including the presence of novel alleles in the class I HLA loci suggest a functionally significant, more rapid evolution of class I compared to class II loci in this South American isolated population.

Alleles↗
PubMed1998

Serology versus ARMS-PCR in prospective HLA-class I typing for bone marrow transplantation.

While HLA class II alleles identification by means of complement mediated lymphocytotoxicity (serology) is almost replaced by DNA typing techniques, serology is still widely used for routine class I typing. The aim of this prospective study was to compare PCR-based Amplification Refractory Mutation System with serology in clinical HLA class I alleles assignment in patients receiving marrow transplants and their potential donors. The total discrepancy rate in 114 consecutively typed individuals for HLA-A and HLA-C alleles was only in favor of ARMS-PCR, whereas HLA-B typing was discrepant also in favor of serology. The discrepancies were higher in patients, particularly in those with acute lymphoblastic leukaemia, than in healthy individuals. We conclude, that ARMS-PCR is clearly superior to serology in definition of class I alleles, which might be of clinical importance particularly for bone marrow transplantation.

Adolescent↗
PubMed1984

Both alpha and beta chains of HLA-DC class II histocompatibility antigens display extensive polymorphism in their amino-terminal domains.

At least three class II antigens, all composed of an alpha and a beta subunit, are encoded in the human major histocompatibility complex, i.e., DR, DC and SB. Two cDNA clones, encoding a DC alpha and a DC beta chain, respectively, were isolated from a cDNA library of the lymphoblastoid cell line Raji (DR3,w6). The two polypeptides predicted from the nucleotide sequences of these clones are each composed of a signal peptide, two extracellular domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Comparison of the DC alpha sequence with two previously published partial sequences shows that the majority of the differences is located in the amino-terminal domain. The differences are not randomly distributed; a cluster of replacements is present in the central portion of the amino-terminal domain. Likewise, the allelic polymorphism of the DC beta chains occurs preferentially in the amino-terminal domain, where three minor clusters of replacements can be discerned. The non-random distribution of the variability of DC alpha and beta chains may be due to phenotypic selection against replacement substitutions in the second domains of the polypeptides.

Amino Acid Sequence↗
PubMed2025

Substitution of Glutamic Acid at Position 71 of DRβ1*04:01 and Collagen-Specific Tolerance Without Alloreactivity.

OBJECTIVE: The DRB1 locus is strongly associated with both susceptibility and resistance to rheumatoid arthritis (RA). DRB1 alleles encoding the VKA or VRA epitope in positions 11, 71, and 74 confer the highest risk of developing RA, whereas the allele encoding VEA is protective. We therefore investigated the feasibility of creating antigen-specific tolerance without inducing alloreactivity by replacing lysine with glutamic acid at position 71 in DRβ1*04:01. METHODS: Individual DRB1 alleles and the DRB1*04:01K71E allele were cloned into T2 cell lines to measure binding of biotinylated peptides. Transgenic animals expressing DRB1*04:01, DRB1*01:01, or DRB1*04:01K71E were injected with collagen to measure T cell proliferation. Skin and bone marrow transplants between DRB1*04:01K71E and DRB1*04:01 mice were performed to determine if the single amino acid change at position 71 would be recognized as foreign. DRB1*04:01 mice transplanted with DRB1*04:01K71E bone marrow were injected with collagen to test if resistance to collagen sensitization could be transferred. RESULTS: Replacing lysine (K) at position 71 in DRβ1*04:01 with glutamic acid (E) blocked collagen peptide binding and rendered the DRB1*04:01K71E mice resistant to collagen sensitization. Skin and bone marrow transplants from DRB1*04:01K71E mice were not rejected by DRB1*04:01 mice, suggesting the single E71 difference was not recognized as allogeneic. Bone marrow from DRB1*04:01K71E mice adoptively transferred antigen-specific tolerance to collagen to DRB1*04:01 mice. CONCLUSION: These studies demonstrate that editing a single amino acid in DRβ1*04:01 blocks collagen peptide binding without inducing alloreactivity and could therefore represent a gene therapy approach to induce antigen-specific passive tolerance.

Animals↗
PubMed1990

Allelic diversification at the class II DQB locus of the mammalian major histocompatibility complex.

The allelic diversity at HLA class II loci either arose after the divergence of hominoid lineages or, alternatively, the polymorphism was present before speciation and has been maintained by selection. Here, we report the use of oligonucleotide primers to amplify, by the polymerase chain reaction, and sequence the polymorphic second exon of the DQB locus from 11 species, spanning more than 40 million years of mammalian evolution. Phylogenetic analysis reveals that of the four human DQB allelic types (DQB1-B4), three (DQB1-3) were found in chimpanzee and gorilla and two (DQB3 and -4) were identified in the rhesus monkey, suggesting that some of these types are 5-20 million years old. The ratio of replacement to silent substitutions was calculated between members of the same allelic type from different species. These results suggest that the evolution of the DQB3 allelic type is more constrained than that of the DQB1 allelic type; both evolve more slowly than the DXB locus, a linked but presumably nonexpressed locus. Further, the clustering of allelic subtypes by species in the phylogenetic tree indicates that allelic diversification has occurred subsequent to the divergence of hominoids. Finally, some haplotype combinations of DQA and DQB alleles are common to several hominoid species and may have been maintained for at least 5 million years.

Alleles↗
PubMed2004

Identification of a new mouse Cd1d2 allele.

The mouse CD1 system is formed by two closely related genes named Cd1d1 and Cd1d2. Cd1d1 encodes a molecule that presents antigens to NKT cells. The function of the Cd1d2 gene has not been elucidated. Here we described a method to analyse variations in mouse Cd1 genes. We found a new allele for Cd1d2 characterized by a point mutation, resulting in a replacement of alanine at position 176 by a valine.

Alanine↗
PubMed1996

Procyclic Trypanosoma brucei cell lines deficient in ornithine decarboxylase activity.

Ornithine decarboxylase (ODC) is a rate limiting enzyme in the biosynthesis of polyamines. We report here the construction of ODC gene deficient Trypanosoma brucei brucei cell lines by homologous recombination and disruption of the two alleles of the ODC gene. With our first stable transfection vector, we replaced the 2.8 kb SacII ODC gene-containing fragment with a hygromycin-B-phosphotransferase gene (hph) cassette transcribed under the control of the endogenous promoter. For the second ODC allele knock-out, we stably transfected similar constructs that contained either the phleomycin or G418 resistance gene cassette, and included 1 mM putrescine in the media. These experiments resulted in two separate ODC- lines: one hygromycin and phleomycin resistant, the other hygromycin and G418 resistant. The two ODC gene knockout lines were verified by Southern and Northern hybridization, and confirmed by Western blot and enzymatic activity assay. There is no ODC expression in the two ODC- lines and the ODC messages in the single ODC gene knockouts were only half of that of the wild type. When grown in the presence of putrescine, the ODC- lines showed little difference, morphologically, from wild type trypanosomes. The growth rate of these lines varied greatly, depending on the concentration of the putrescine. Interestingly, when putrescine was completely withdrawn from the media, the ODC- trypanosomes soon reached a plateau phase and some cells remained viable for 7-8 weeks. The starved cells could be rescued by the addition of putrescine or introducing back the ODC gene. Cell cycle analysis suggested that putrescine is required for G1-S transition in the procyclic form T. brucei.

Alleles↗
PubMed2000

Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C.

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.

3' Untranslated Regions↗
PubMed2004

Efficient DNA cassette exchange in mouse embryonic stem cells by staggered positive-negative selection.

Recombinase-mediated cassette exchange (RMCE), when applied to mouse embryonic stem (ES) cells, promises to increase the ease with which genetic alterations can be introduced into targeted genomic loci in the mouse. However, existing selection strategies for identifying ES cells in which replacement DNA cassettes from a carrier plasmid have been exchanged correctly into a defined locus are suboptimal. Here, we report the generation in mouse ES cells of a loxed cassette acceptor (LCA) allele within the glucokinase (gk) gene locus. Using the gkLCA as a test allele, we developed a staggered positive-negative selection strategy that facilitates efficient identification of ES cell clones in which a DNA replacement cassette from a carrier plasmid has been exchanged correctly into the gkLCA allele. This selection strategy, by facilitating more efficient production of ES cell clones with various replacement DNA cassettes, should accelerate targeted repetitive introduction of gene modifications into the mouse.

Alleles↗
PubMed1979

Maximum geographic range of a mutant allele considered as a subtype of a Brownian branching random field.

A rare allelic type is modeled as a field of individuals diffusing independently in d-dimensional space (d = 1,2,...), in which individuals are replaced by random numbers of offspring at a constant rate. In an infinite-allele model with selectively identical alleles, the offspring distribution would have mean 1 - u, in which u is the mutation rate; otherwise mean (1 - u)w, in which w is the relative fitness of the allele. Let p(x) be the probability that some descendent of an individual initially at zero diffuses unilaterally as far as x(d = 1), or else the probability that some descendent of an individual at x diffuses within a > 0 of the origin (d >/= 2). A nonlinear differential equation is found for p(x) that is solvable for d = 1 and related to Emden's equation for d >/= 2. For p(x) >/= 10(-4) and u </= 10(-5), genetic drift is more important than mutation in the behavior of p(x) (d = 1). If u = 0 and w = 1, p(x) approximately C/x(2) as x --> infinity for d </= 3. As a mathematical application, it is shown that if the initial distribution is uniform Poisson, a bounded open set K is visited by individuals in the field at arbitrarily large times if d >/= 2 but not if d = 1.

Journal Article↗
PubMed1989

Finnish type of low density lipoprotein receptor gene mutation (FH-Helsinki) deletes exons encoding the carboxy-terminal part of the receptor and creates an internalization-defective phenotype.

A specific type of gene mutation affecting the LDL receptor has been found in many Finnish patients with familial hypercholesterolemia (FH). The mutant allele is characterized by a 9.5-kb deletion extending from intron 15 to exon 18. Molecular cloning and sequencing of a cDNA segment corresponding to the deleted allele indicated that the mutant receptor differs radically from the normal one because of loss of the domains encoded by exons 16, 17, and 18. The carboxy-terminal portion of the normal receptor, comprising the amino acids 750-839, has been replaced by an unrelated stretch of 55 amino acids. The mutant allele was found to occur in 23 (50%) of 46 unrelated FH patients with an established functional defect in the LDL receptor. In cultured fibroblasts from the FH patients with the 9.5-kb deletion, both receptor-mediated binding and internalization of 125I-LDL were lower than normal, the former, on average, by 25%, and the latter, on average, by 50%. This combined functional defect probably results from both impaired attachment and impaired internalization of the mutated receptor. It remains to be investigated whether this Finnish type of LDL receptor gene mutation, here designated FH-Helsinki, occurs in other ethnic groups.

Adult↗
PubMed2001

A novel HLA-DRB1 sequence, DRB1*11272.

We describe the complete exon 2 sequence of a novel HLA-DRB1 allele, DRB1*11272. This allele differs from the DRB1*11271 allele by a synonymous mutation in codon 77 where an AAT is replaced with AAC, both encode for the amino acid asparagine. The same motif at codon 77 has also been found in DRB1*1107, DRB1*1333, DRB1*0422 and in most DRB1*03 alleles. A partial exon 2 sequence of this allele has previously been deposited in the EMBL Sequence Database under the accession number AF186407.

Alleles↗