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PubMed2006

Attenuated Bordetella pertussis: new live vaccines for intranasal immunisation.

Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious pathogen with a strong capacity to colonize the human respiratory tract. A single infection with virulent B. pertussis induces strong mucosal and systemic humoral and cellular immune responses, as well as long-lasting protection in humans. Therefore, B. pertussis may be a suitable vector for intranasal vaccination against a variety of diseases by a single dose. We showed that intranasally administered recombinant live B. pertussis expressing heterologous antigens induce mucosal immune responses in the respiratory and the genital tracts of mice as well as systemic antibody responses. To consider live B. pertussis for human vaccination, we, therefore, aimed at the development of attenuated strain. Allelic exchange was used to delete the dermonecrotic toxin gene and to replace the pertussis toxin gene by an allele encoding inactive toxin. To reduce the production of tracheal cytotoxin, the ampG gene was over-expressed. After various rounds of homologous recombination, B. pertussis BPZE, affected in the production or activity of three major toxins, was isolated. Histological examinations of infected mice confirmed the strong attenuation of this strain. Nevertheless, it colonized the mouse respiratory tract over a period of roughly 1 month. The level of protection against subsequent challenge with virulent wild-type B. pertussis, induced by a single intranasal administration of BPZE was at least as high as that induced by two injections of commercial acellular vaccine. We conclude that live attenuated B. pertussis may be an attractive vaccine candidate to be administered in a single intranasal dose against whooping cough. Moreover, live recombinant B. pertussis may be used as a vector for intranasal vaccination against a variety of diseases.

Administration, Intranasal↗
PubMed1983

Isolation and properties of strains of Micrococcus (Deinococcus) radiodurans unable to excise ultraviolet light-induced pyrimidine dimers from DNA: evidence for two excision pathways.

A mutant of Deinococcus (formerly Micrococcus) radiodurans (strain 302, mutant in mtcA) sensitive to both the lethal effect of mitomycin C and the mutagenic effect of simple alkylating agents, but having wild-type resistance to UV light, was treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine in an attempt to isolate strains deficient in the ability to excise UV-induced pyrimidine dimers. Three strains were isolated that were UV-sensitive, but had wild-type resistance to the lethal effect of methyl methanesulphonate and all were shown to be unable to excise pyrimidine dimers. The three strains UVS9, UVS25 and UVS78 had, in addition to the mutation in mtcA, mutations in loci designated uvsC, uvsD and uvsE, respectively. When the mutant mtcA gene was replaced by its wild-type allele in all three strains they became UV- and mitomycin C-resistant. On incubating the double mutants UVS9, UVS25 and UVS78 with wild-type DNA about 50% of the transformants selected for UV resistance were mitomycin C-sensitive and about 50% resistant depending on whether the mutant mtcA or the uvsC, D or E genes had been replaced by their wild-type alleles. Although strains mutant singly in uvsC, D or E were UV-resistant the rates of excision of pyrimidine dimers differed between them and was slower in all of them than in the wild-type and strain 302. The results indicate that wild-type D. radiodurans possesses two pathways for the excision of pyrimidine dimers and that mutational blocks in both must exist for the excisionless phenotype to be expressed.

DNA Repair↗
PubMed2005

Identification and characterization of a novel allele of Escherichia coli dnaB helicase that compromises the stability of plasmid P1.

Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34 degrees C. Shifting dnaB277 cells to 42 degrees C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.

Adenosine Triphosphatases↗
PubMed2002

HPA-1a phenotype-genotype discrepancy reveals a naturally occurring Arg93Gln substitution in the platelet beta 3 integrin that disrupts the HPA-1a epitope.

A single nucleotide polymorphism (SNP) at position 196 in the beta 3 integrin causes a Leu33Pro substitution in the mature protein. Alloimmunization against the beta 3Leu33 form (human platelet antigen [HPA]-1a, Pl(A1), Zw(a)) in patients who are beta 3Pro33 homozygous (HPA-1b1b, Pl(A2A2), Zw(bb)) causes neonatal alloimmune thrombocytopenia, posttransfusion purpura, or refractoriness to platelet transfusion. Studies with recombinant proteins have demonstrated that amino acids 1 to 66 and 288 to 490 of the beta 3 integrin contribute to HPA-1a epitope formation. In determining the HPA-1a status of more than 6000 donors, we identified a donor with an HPA-1a(weak) phenotype and an HPA-1a1b genotype. The platelets from this donor had normal levels of surface alpha IIb beta 3 but reacted only weakly with monoclonal and polyclonal anti-HPA-1a by whole blood enzyme-linked immunosorbent assay (ELISA), flow cytometry, and sandwich ELISA. We reasoned that an alteration in the primary nucleotide sequence of the beta 3Leu33 allele of this donor was disrupting the HPA-1a epitope. In agreement with this hypothesis, sequencing platelet RNA-derived alpha IIb and beta 3 cDNA identified a novel G/A SNP at position 376 of the beta 3 integrin that encodes for an Arg93Gln replacement in the beta 3Leu33 allele. Coexpression of the beta 3Leu33Gln93 encoding cDNA in Chinese hamster ovary cells with human alpha IIb cDNA showed that the surface-expressed alpha IIb beta 3 reacted normally with beta 3 integrin-specific monoclonal antibodies but only weakly with monoclonal anti-HPA-1a. Our results show that an Arg93Gln mutation in the beta 3Leu33 encoding allele disrupts the HPA-1a epitope, suggesting that Arg93 contributes to the formation of the HPA-1a B-cell epitope.

Amino Acid Substitution↗
PubMed1997

Functional analysis of human RPS14 null alleles.

Previously we described a large collection of cloned human DNAs that encode chemically defined missense mutations within the ribosomal protein S14 sequence. We determined that biologically inactive (i.e. null) alleles resulted primarily from point mutations targeted to two internal segments of the S14-coding sequence and designated these functionally critical regions as domains B and D. Further, we inferred that structural determinants within domains B and D are required for proper incorporation of the S14 protein into nascent 40 S ribosomal particles and/or for the normal function of mature cytoplasmic ribosomes. In this study we have used immunofluorescence to monitor the intracellular trafficking of epitopically labeled human S14 protein isoforms transiently expressed by cultured Chinese hamster cells. Data obtained distinguish null alleles of RPS14 which encode proteins that are not incorporated into pre-ribosomal subunit particles from null alleles whose products are compatible with normal ribosome assembly processes but result in functionally inactive cytoplasmic 40 S ribosomal subunits. Mutations assigned to the first allele class involve amino acid replacements located within S14 domains B and D; whereas mutations assigned to the second class are distributed throughout the S14 protein-coding sequence.

Alleles↗
PubMed2004

Positive selection at sites of multiple amino acid replacements since rat-mouse divergence.

New alleles become fixed owing to random drift of nearly neutral mutations or to positive selection of substantially advantageous mutations. After decades of debate, the fraction of fixations driven by selection remains uncertain. Within 9,390 genes, we analysed 28,196 codons at which rat and mouse differ from each other at two nucleotide sites and 1,982 codons with three differences. At codons where rat-mouse divergence involved two non-synonymous substitutions, both of them occurred in the same lineage, either rat or mouse, in 64% of cases; however, independent substitutions would occur in the same lineage with a probability of only 50%. All three non-synonymous substitutions occurred in the same lineage for 46% of codons, instead of the 25% expected. Furthermore, comparison of 12 pairs of prokaryotic genomes also shows clumping of multiple non-synonymous substitutions in the same lineage. This pattern cannot be explained by correlated mutation or episodes of relaxed negative selection, but instead indicates that positive selection acts at many sites of rapid, successive amino acid replacement.

Alleles↗
PubMed1981

Deletion analysis of the tumorous-head (tuh-3) gene in Drosophila melanogaster.

In the presence of the naturally occurring maternal-effect alleles tuh-1h or tuh-1g, the tuh-3 mutant gene can cause the tumorous-head trait or the sac-testis trait. The tuh-3 gene functions as a semidominant in the presence of the tuh-1h maternal effect. Eye-antennal structures are replaced by posterior abdominal tergites and genital structures. If tuh-1h is replaced by its naturally occurring allele tuh-1g, tuh-3 functions as a recessive hypomorph and the defect switches from anterior to posterior structures, with a male genital-disc defect appearing with variable penetrance. Function and regulation of tuh-3+ may better be understood in light of the cytological localization of tuh-3 either adjacent to or as part of the bithorax complex. The tuh-3+ gene product appears to be essential for normal development, at least in the posterior end of the embryo.

Alleles↗
PubMed2003

The low-frequency allele of the platelet collagen signaling receptor glycoprotein VI is associated with reduced functional responses and expression.

Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P <.0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P <.001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer-100 (t test, P =.065 and P =.061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.

Adult↗
PubMed1992

Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein.

A methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing-over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high-frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.

Acyl Carrier Protein↗
PubMed2004

LFIRE-1/HFREP-1, a liver-specific gene, is frequently downregulated and has growth suppressor activity in hepatocellular carcinoma.

We have previously identified several novel genes, which are differentially expressed among human normal liver and hepatocellular carcinomas (HCCs). The full-length liver fibrinogen-related gene-1 (LFIRE-1) cDNA was cloned from the human normal liver cDNA library. LFIRE-1 is highly homologous to HFREP-1 with discrepancy at 5' untranslated region (UTR) and encodes the same fibrinogen-related protein, which suggest that these two sequences might be alternative splicing forms of the same gene, LFIRE-1/HFREP-1, located at human chromosome 8p22. The LFIRE-1 and HFREP-1 are specifically expressed in normal human liver tissue, but reduced or undetectable in most of HCC specimens at both RNA and protein level. Furthermore, the reduction or nonexpression of LFIRE-1/HFREP-1 is significantly associated with the degree of tumor differentiation. Loss of heterozygosity (LOH) analysis revealed allelic loss of LFIRE-1/HFREP-1 on chromosome 8p22 in 57.1% (24/42) of HCC specimens. We detected three inactivation mutations among 45 cases of HCC specimens examined, two of which lost the remaining allele and the third had a replacement of conserved cysteine residue with glycine residue. Notably, the downregulation of LFIRE-1/HFREP-1 expression is frequently associated with allelic loss. The reduction of LFIRE-1/HFREP-1 expression by antisense approach enhances cancer cell proliferation and colony formation in soft agar. Moreover, restoration of exogenous wild-type LFIRE-1/HFREP-1 expression but not LFIRE-1/HFREP-1 missense mutations in human HCC cells inhibited their anchorage-dependent or -independent growth in vitro, and suppressed their tumorigenicity in nude mice. In conclusion, our data demonstrated that liver-specific gene LFIRE-1/HFREP-1 was frequently downregulated and might possess growth suppressor activity in HCC.

5' Untranslated Regions↗
PubMed2003

L-proline accumulation and freeze tolerance of Saccharomyces cerevisiae are caused by a mutation in the PRO1 gene encoding gamma-glutamyl kinase.

We previously isolated a mutant which showed a high tolerance to freezing that correlated with higher levels of intracellular L-proline derived from L-proline analogue-resistant mutants. The mutation responsible for the analogue resistance and L-proline accumulation was a single nuclear dominant mutation. By introducing the mutant-derived genomic library into a non-L-proline-utilizing strain, the mutant was found to carry an allele of the wild-type PRO1 gene encoding gamma-glutamyl kinase, which resulted in a single amino acid replacement; Asp (GAC) at position 154 was replaced by Asn (AAC). Interestingly, the allele of PRO1 was shown to enhance the activities of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase, both of which catalyze the first two steps of L-proline synthesis from L-glutamate and which together may form a complex in vivo. When cultured in liquid minimal medium, yeast cells expressing the mutated gamma-glutamyl kinase were found to accumulate intracellular L-proline and showed a prominent increase in cell viability after freezing at -20 degrees C compared to the viability of cells harboring the wild-type PRO1 gene. These results suggest that the altered gamma-glutamyl kinase results in stabilization of the complex or has an indirect effect on gamma-glutamyl phosphate reductase activity, which leads to an increase in L-proline production in Saccharomyces cerevisiae. The approach described in this paper could be a practical method for breeding novel freeze-tolerant yeast strains.

Amino Acid Sequence↗
PubMed2003

Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij.

Defects in the assembly of dolichol-linked oligosaccharide or its transfer to proteins result in severe, multi-system human diseases called Type I congenital disorders of glycosylation. We have identified a novel CDG type, CDG-Ij, resulting from deficiency in UDP-GlcNAc: dolichol phosphate N-acetyl-glucosamine-1 phosphate transferase (GPT) activity encoded by DPAGT1. The patient presents with severe hypotonia, medically intractable seizures, mental retardation, microcephaly, and exotropia. Metabolic labeling of cultured dermal fibroblasts from the patient with [2-(3)H]-mannose revealed lowered incorporation of radiolabel into full-length dolichol-linked oligosaccharides and glycoproteins. In vitro enzymatic analysis of microsomal fractions from the cultured cells indicated that oligosaccharyltransferase activity is normal, but the GPT activity is reduced to approximately 10% of normal levels while parents have heterozygous levels. The patient's paternal DPAGT1 allele contains a point mutation (660A>G) that replaces a highly conserved tyrosine with a cysteine (Y170C). The paternal allele cDNA produces a full-length protein with almost no activity when over-expressed in CHO cells. The maternal allele makes only about 12% normal mature mRNA, while the remainder shows a complex exon skipping pattern that shifts the reading frame encoding a truncated non-functional GPT protein. Thus, we conclude that the DPAGT1 gene defects are responsible for the CDG symptoms in this patient. Hum Mutat 22:144-150, 2003.

Alleles↗
PubMed2005

Two-step method for constructing unmarked insertions, deletions and allele substitutions in the yeast genome.

We describe three extensions of the method of site-specific genomic (SSG) mutagenesis. These three extensions of SSG mutagenesis were used to generate precise insertion, deletion, and allele substitution mutations in the genome of the budding yeast, Saccharomyces cerevisiae. These mutations are termed precise because no attached sequences (e.g., marker genes or recombination sites) are retained once the method is complete. Because the method is PCR-based, neither DNA cloning nor synthesis of long oligonucleotides is required. We demonstrated the efficacy of these methods by deleting an ORF, inserting the tandem affinity purification (TAP) tag, and replacing a wild-type allele with a mutant allele.

Alleles↗
PubMed2004

Influence of genetic variations of ethanol-metabolizing enzymes on phenotypes of alcohol-related disorders.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase-2 (ALDH2) play central roles in the metabolism of ethanol and its metabolite, acetaldehyde, in the liver. In ADH2, one nucleotide replacement causes either a super-active beta 2 subunit encoded by the ADH2*2 allele or a less active beta 1 subunit (ADH2*1 allele). In the same way, a G/A replacement at codon 487 of the ALDH2 gene produces an inactive form of the enzyme. Because the geno-types of these genes may explain individual differences in concentration and elimination of ethanol and acetaldehyde in the blood after drinking, they could be used as models to elucidate the contribution of these substances to the development of addiction and various types of organ damage. We have examined the influence of genetic variations of these enzymes on alcohol-related disorders in the Japanese. The results revealed that (1) the less active allele of the ADH2 gene (ADH2*1) is associated with an increased risk for alcohol dependence, alcohol-induced persistent amnestic disorder, alcohol withdrawal syndrome, and cancer of the upper GI tract; (2) the inactive allele of the ALDH2 gene (ALDH2*2) is associated with a decreased risk for alcohol dependence, and an increased risk for alcoholic polyneuropathy and cancer in the same region; and (3) these genetic variations modify clinical features of alcohol dependence. Possible mechanisms of altered risk for these disorders are discussed.

Alcohol Dehydrogenase↗
PubMed1989

Diversity and diversification of HLA-A,B,C alleles.

The nucleotide sequences encoding 14 HLA-A,B,C and 5 ChLA-A,B,C molecules have been determined. Combining these sequences with published data has enabled the polymorphism in 40 HLA-A,B,C and 9 ChLA-A,B,C alleles to be analyzed. Diversity is generated through assortment of point mutations by recombinational mechanisms including gene and allelic conversions. The distribution and frequency of silent and replacement substitutions indicate that there has been positive selection for allelic diversity in the 5' part of the gene (exons 1 to 3) and for allelic homogenization and locus specificity in the 3' part of the gene (exons 4 to 8). These differences may correlate with the lengths of converted sequences in the two parts of the gene and frequency of the CpG dinucleotide. Locus-specific divergence of HLA-A,B, and C demonstrates that recombinational events involving alleles of a locus have been more important than conversion between loci. This contrasts with the predominance of gene conversion events in the evolution of mutants of the H-2Kb gene. However, a striking example of gene conversion involving HLA-B and C alleles of an oriental haplotype has been found. Comparison of human and chimpanzee alleles reveals extensive sharing of polymorphisms, confirming that diversification is a slow process, and that much of contemporary polymorphism originated in ancestral primate species before the emergence of Homo sapiens. There is less polymorphism at the HLA-A locus compared to HLA-B, with greater similarity also being seen between HLA-A and ChLA-A alleles than between HLA-B and ChLA-B alleles. Although greater diversity is seen in the 5' "variable" exons of HLA-B compared to HLA-A, there is increased heterogeneity in the 3' "conserved" exons of HLA-A compared to HLA-B.

Alleles↗
PubMed2006

Routine HLA sequence-specific-oligonucleotide typing and confirming sequence-based typing of a prospective bone marrow donor identified a novel B*27 allele, HLA-B*2734.

In this study, we report the identification of a new human leukocyte antigen-B allele in a sample that was tested in our routine typing for volunteer bone marrow donors. This novel allele officially named B*2734 was found in a female donor of Caucasoid origin (laboratory code 158567). The search for unrelated bone marrow donors was initiated by the Aktion Knochenmarkspende Bayern. In comparison with the closest related allele B*2707, B*2734 differs at two nucleotide positions, all located in exon 3, at position 412, 'A' in B*2707 is replaced by 'G' and at position 486, 'G' is replaced by 'A' in the novel allele. The single nucleotide polymorphism at position 412 results in an amino acid substitution at codon 138. The amino acid asparagine is changed to aspartic acid in B*2734 allele. The second nucleotide exchange is a silent mutation and codon 163 remains unaffected as ACG-->ACA, still codes for alanine.

Alleles↗
PubMed2001

Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants.

In many higher plants, cellulose synthesis is inhibited by isoxaben and thiazolidinone herbicides such as 5-tert-butyl-carbamoyloxy-3-(3-trifluromethyl) phenyl-4-thiazolidinone. Semidominant mutations at the IXR1 and IXR2 loci of Arabidopsis confer isoxaben and thiazolidinone resistance. Isolation of the IXR1 gene by map-based cloning revealed that it encodes the AtCESA3 isoform of cellulose synthase. The two known mutant alleles contain point mutations that replace glycine 998 with aspartic acid, and threonine 942 with isoleucine, respectively. The mutations occur in a highly conserved region of the enzyme near the carboxyl terminus that is well separated from the proposed active site. Although the IXR1 gene is expressed in the same cells as the structurally related RSW1 (AtCESA1) cellulose synthase gene, these two CESA genes are not functionally redundant.

Alleles↗
PubMed1993

P-element-induced interallelic gene conversion of insertions and deletions in Drosophila melanogaster.

We studied the process by which whd, a P-element insertion allele of the Drosophila melanogaster white locus, is replaced by its homolog in the presence of transposase. These events are interpreted as the result of double-strand gap repair following excision of the P transposon in whd. We used a series of alleles derived from whd through P-element mobility as templates for this repair. One group of alleles, referred to collectively as whd-F, carried fragments of the P element that had lost some of the sequences needed in cis for mobility. The other group, whd-D, had lost all of the P insert and had some of the flanking DNA from white deleted. The average replacement frequencies were 43% for whd-F alleles and 7% for the whd-D alleles. Some of the former were converted at frequencies exceeding 50%. Our data suggest that the high conversion frequencies for the whd-F templates can be attributed at least in part to an elevated efficiency of repair of unexpanded gaps that is possibly caused by the closer match between whd-F sequences and the unexpanded gap endpoints. In addition, we found that the gene substitutions were almost exclusively in the direction of whd being replaced by the whd-F or whd-D allele rather than the reverse. The template alleles were usually unaltered in the process. This asymmetry implies that the conversion process is unidirectional and that the P fragments are not good substrates for P-element transposase. Our results help elucidate a highly efficient double-strand gap repair mechanism in D. melanogaster that can also be used for gene replacement procedures involving insertions and deletions. They also help explain the rapid spread of P elements in populations.

Alleles↗