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PubMed1994

Mutations in the structural gene for cytochrome c result in deficiency of both cytochromes aa3 and c in Neurospora crassa.

The cyt-12-12 mutant of Neurospora crassa is characterized by slow growth and a deficiency of spectrophotometrically-detectable cytochromes aa3 and c. Using a sib-selection procedure we have isolated the cyt-12+ allele from a cosmid library of N. crassa genomic DNA. Characterization of the cyt-12+ allele reveals that it encodes the structural gene for cytochrome c. DNA sequence analysis of the cyt-12-12 allele revealed a mutation in the cytochrome c coding sequence that results in replacement of a glycine residue, which is invariant in the cytochrome c of other species, with an aspartic acid. Genetic analysis confirms that cyt-12-12 is allelic with the previously-characterized cyc-1-1 mutant, which was also shown to affect the single locus encoding cytochrome c in N. crassa. We suggest that the amount of functional cytochrome c present in mitochondria influences the level of cytochrome aa3.

Alleles↗
PubMed2000

Identification by sequencing based typing and complete coding region analysis of three new HLA class II alleles: DRB3*0210, DRB3*0211 and DQB1*0310.

The study of HLA class II polymorphism by direct exon 2 DNA sequencing analysis has been established to be a reliable and accurate high-resolution typing procedure. This approach shows some advantages in relation to previous methods, polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and sequence-specific primers (PCR-SSP), basically due to the capability of analysis for the complete sequenced genomic region, including non-polymorphic motifs. DRB3 and DQB1 sequencing based typing (SBT) in unrelated bone marrow donor searching allowed us to detect three new alleles. The complete coding region sequences were characterised from cDNA. Two new DRB3 alleles, DRB3*0210 and DRB3*0211, were described in two Caucasian bone marrow donors. Both sequences showed single point mutations regarding DRB3*0202, producing amino acid replacements at positions 51 (Asp to Thr) and 67 (Leu to Ile), respectively. These two point mutations can be found in other DRB alleles, and suggest that gene conversion would be involved in the origin of both alleles. A new DQB1 sequence was found in a Spanish patient that showed two nucleotide differences, positions 134 and 141, with regard to its close similar DQB1*03011 allele. Only substitution at position 134 provoked amino acid replacement at residue 45, Glu to Gly. This single amino acid change would be involved in the lack of serologic recognition of this new molecule by DQ7-specific reagents.

Alleles↗
PubMed1983

Evolution of immunoglobulin V genes: evidence indicating that recently duplicated human V kappa sequences have diverged by gene conversion.

We have analyzed several closely related members of the gene family encoding the variable regions of human immunoglobulin kappa light chains (V kappa genes). Two of these genes differ at a single nucleotide out of 940 bases sequenced, and are believed to be alleles of a locus called HK 101. This substitution results in an amino acid replacement in the first complementarity-determining region of the kappa chain. We also compared the structures of two nonallelic human V kappa loci (HK 101 and HK 137) and found a high degree of sequence homology over a region at least 13.5 kb long. This long block of homology indicates that these two loci arose from a recent gene duplication. The DNA sequences of these two nonallelic V kappa genes exhibit a very unusual distribution of nucleotide substitutions. Seven of the ten substitutions found among 940 bases are clustered in a 39 base stretch encoding the first complementarity-determining region and the second framework region of the protein. We suggest that this cluster of substitutions was generated by a gene conversion in which a small segment of one gene was replaced with the homologous segment from another V kappa gene.

Alleles↗
PubMed2004

A novel cis-AB allele derived from the A transferase gene by nucleotide substitution C796A.

The cis-AB is a very rare phenotype in the ABO blood group system. It corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B antigens. Until now, gene sequences of only 3 cis-AB alleles were characterized. One was the A(1v) allele with a nucleotide substitution G803C at codon 268; the second was the B allele with a nucleotide substitution A796C at codon 266; and the third arose from a point mutation C700T at codon 234 in exon 7 of the B transferase gene. In this study, we found a novel cis-AB allele when performing paternity tests in Chang Gung Memorial Hospital in Taiwan. Although his father was O blood type, a serologically AB blood type child was confirmed as being his father's offspring on the basis of 16 microsatellite markers (99.97% plausibility for the child and father). Exons 6 and 7 of the child's ABO alleles were characterized by direct sequencing and gene cloning. The results showed that the child has one O(1) allele and the second allele is almost identical to A(1*02) allele except for a single point mutation at nucleotide position 796, where an A replaces a C and leads to a change of leucine to methionine at amino acid 266. This implies that the child's O(1) allele was inherited from his father and the other allele was inherited from his mother. In conclusion, the novel cis-AB allele reported here is derived from the A transferase gene through a nucleotide substitution C796A, which differs from the 3 previously reported cis-AB alleles.

ABO Blood-Group System↗
PubMed1995

Suppression of recJ exonuclease mutants of Escherichia coli by alterations in DNA helicases II (uvrD) and IV (helD).

The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, "uvrD517am," at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517am mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517am as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517am derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor "srjD" is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ.

Adenosine Triphosphatases↗
PubMed1991

Sequencing of the variant thyroxine-binding globulin (TBG)-Quebec reveals two nucleotide substitutions.

Thyroxine-binding globulin (TBG) is a liver glycoprotein that transports thyroid hormone in serum. In 1987 a variant TBG was discovered in an infant born in Quebec, following an investigation prompted by the finding of low blood thyroxine (T4) level on screening for neonatal hypothyroidism. This variant, TBG-Quebec, has cathodal shift on isoelectric focusing, reduced affinity for thyroxine, and markedly reduced stability. The latter property of the variant molecule is probably responsible for the partial TBG deficiency. We now report the results of sequencing of the entire coding region and exon-intron junctions of TBG-Quebec, which revealed two nucleotide substitutions; one, located in exon 3, changes the normal codon 283 of TTG (leucine) to that of TTT (phenylalanine), and the other, in exon 4, results in the replacement of the normal histidine-331 (CAT) by tyrosine (TAT). Allele-specific amplification (ASA) confirmed the cosegregation of the two nucleotide substitutions with the TBG-Quebec phenotype in individual members of this family. The substitution in codon 283, but not that in codon 331, has been previously described and, when occurring alone, does not alter the properties of the gene product. Thus, it appears that the replacement of histidine-331 by tyrosine is responsible for the observed altered properties of TBG-Quebec. However, the question of whether substitution of both amino acids is necessary for expression of the variant phenotype has yet to be answered.

Alleles↗
PubMed1991

Sequence of the variant thyroxine-binding globulin (TBG) in a Montreal family with partial TBG deficiency.

The variant thyroxine-binding globulin in a family from Montreal (TBG-M) has a reduced affinity for thyroxine, shows a slight cathodal shift on isoelectric focusing, and has an increased susceptibility to inactivation by heat and acid. We present the molecular basis for TBG-M, deduced by sequencing the entire 1245-bp coding regions and intron/exon junctions of the TBG gene of an affected hemizygous male. A single nucleotide substitution in the codon for amino acid 113 of the mature protein (GCC to CCC) was found, resulting in the replacement of alanine by proline. The mutation was confirmed by allele-specific amplification of genomic DNA from the propositus and three other affected family members. Since point mutations throughout the molecule have been shown to alter the properties of variant TBGs, and because amino acid substitutions with proline are known to impair stability and function of proteins, the replacement of alanine 113 by proline provides a logical explanation for the observed properties of TBG-M.

Amino Acid Sequence↗
PubMed2001

Chitotriosidase genotype and plasma activity in patients type 1 Gaucher's disease and their relatives (carriers and non carriers).

BACKGROUND AND OBJECTIVES: Chitinases are enzymes that hydolyze chitin and have been found in a wide variety of nonvertebrate species; recently an human analogue of chitinases, chitotriosidase (CT) has been identified. Extreme elevations of plasma CT activity are observed in patients with Gaucher disease (GD), being Gaucher cells the source of the CT. It has been reported a 24 bp duplication in CT gene that results an inactive protein. The carrier prevalence is high as 30 to 40% and the CT activity is half that in wild individuals. However no systematic evaluation of plasma CT activity has been carried in GD patients taken into account status of allele defective for CT and dose in patients on enzyme replacement therapy (ERT). DESIGN AND METHODS: We had previously study 210 subjects from 99 unrelated Spanish GD families; 121 were non-affected carriers and 89 were non-carriers to establish carrier prevalence of CT genotypes. Plasma CTactivity and CTgenotypes by PCR and gel electrophoresis have been measured in 109 GD patients before treatment. We also evalued CT activity after ERT with alglucerase in 68 patients. RESULTS: Three patients had defective activities of CT. The carrier prevalence for 24 bp duplication was 35% and the allele frequency 0.20. No correlation between CT activity and GBA genotype was detected and between CT activity and visceral or skeletal disease in GD patients. Untreated affected patients, non-carriers for the duplication, had higher CT activity than carriers (p<.0001). CT activity decreased dramatically during the first 12 months of ERT; even after 3 years of therapy a persistent fall of CT activity was observed. How ever within 3 years of treatment, a significant difference in the mean decrease of CT activity was present among the groups of patients on varying alglucerase doses (p<.01). After 12 months of ERT the activity of plasma CT decline in the same percentage in both groups: heterozygous for the carriers of 24bp duplication and wild type, but thereafter CT activity decline more slowly in carriers than non carriers. INTERPRETATION AND CONCLUSIONS: The present data can be used as a reference to interpret the CT activity in GD patients with or without ERT, as well as to evaluate the doses response and can be used as a reference to interpret the CTactivity in carriers and non-carriers.

Adolescent↗
PubMed1989

Activation of an excluded immunoglobulin allele in a human B lymphoma cell line.

Mature B cells that express surface immunoglobulin (Ig) are usually committed to their original Ig product. It was shown that such a cell can replace its light chain by rearranging and expressing a new light chain from the other allele. Anti-idiotype antibodies were used to isolate idiotypic variants from a surface IgM+lambda+ human B cell tumor line. The variants expressed a new lambda light chain. Both the original and the new lambda transcripts were present in the variant cells, but only the new one was expressed as a protein on the cell surface. Therefore, although the cell exhibited allelic exclusion and had only one Ig receptor at a time, the commitment to a particular light chain gene was reversible.

Alleles↗
PubMed1990

The role of the delta-lysin gene (hld) in the regulation of virulence genes by the accessory gene regulator (agr) in Staphylococcus aureus.

The synthesis of at least 14 extracellular toxins and enzymes in Staphylococcus aureus is regulated by a set of trans-acting elements from the agr (accessory gene regulator) locus. We have shown that the delta-lysin gene (hld) that is transcribed from a promoter immediately upstream of the agr locus, and which is positively controlled by agr, is part of this regulatory system. Deletion replacement mutagenesis of the chromosomal hld gene had the same pleiotropic effect on the synthesis of several virulence factors as agrA mutations. Characteristically, these mutants had an almost complete block in the synthesis of alpha-toxin, serin- and metalloprotease, whereas synthesis of protein A was greater than 10-fold higher than in the parental strain. Corresponding changes in the levels of alpha-toxin and protein A mRNAs were demonstrated by northern blotting experiments. The effects of the hld deletion mutation could be fully complemented by the hld gene on a plasmid. A plasmid insertion mutation in the 3' non-coding region of hld had a similar effect on exoprotein synthesis, indicating a role of the hld transcript in the regulation of exoprotein synthesis. This was confirmed by the finding that the effects of alpha-toxin and protein A synthesis by the hld deletion replacement mutation could be fully complemented by a hld allele in which we had introduced an early stop codon in the delta-lysin structural gene. However, the mutant hld allele could not complement the defect in production of extracellular proteases, indicating that delta-lysin may act in conjunction with its mRNA to regulate the expression of some exoprotein genes.

Amino Acid Sequence↗
PubMed1997

Short tandem repeat typing by capillary array electrophoresis: comparison of sizing accuracy and precision using different buffer systems.

Polymorphic microsatellite markers are widely used in gene discovery and mapping, human identification, agricultural genetics, and diagnosis of triplet-repeat expansion disorders. Reliable genotyping of these markers requires polymerase chain reaction (PCR) amplification and very-high-resolution electrophoresis. Capillary array electrophoresis offers extremely fast, high-resolution separation of DNA and more automated sample processing because labor-intensive slab-gel pouring and sample loading are eliminated. We report a simple, reliable procedure for preparing PCR samples for electrokinetic injection into capillaries using a 96-well tray and float dialysis. We developed an improved sizing standard for genotyping and used it to evaluate systematically the sizing accuracy and precision of low-viscosity, replaceable matrix formulations. Our study sizing over 28,000 alleles yielded an average precision of +/- 0.12 bp for fragments up to 350 bp. Low-viscosity formulations permit low-pressure matrix injection (40 psi) and a turnaround time of 70 min for 48-96 samples.

Alleles↗
PubMed1998

Three murine cataract mutants (Cat2) are defective in different gamma-crystallin genes.

A number of murine cataract mutations have been localized to chromosome 1 close to the gamma-crystallin gene cluster (Cryg) (Everett et al., 1994, Genomics 20: 429-434; Löster et al., 1994, Genomics 23: 240-242). Based on the size of the mapping or allelism tests they have not been shown to be genetically distinct and have been assigned to locus symbol Cat2. Here we assign three mutations to the respective gamma-crystallin gene. Using a systematic candidate gene approach to analyze the entire Cryg cluster, an A-->G transition was found in exon 2 of Cryga for the ENU-436 mutation and is designated Cryga1Neu. The mutant allele Crygbnop (formerly Cat2(nop)) is caused by a replacement of 11 bp by 4 bp in the third exon of Crygb, while a C-->G transversion in exon 3 of Cryge has been found for the Cryget (formerly Cat2(t)) mutation. For the mutation Cryga1Neu, an Asp-->Gly exchange is deduced, whereas the mutations Crygbnop and Cryget lead to the formation of in-frame stop codons and give rise to truncated proteins of 144 and 143 amino acids, respectively. The effects of the mutations upon gamma-crystallin structure are likely to be quite different. The Cryga1Neu mutation is expected to affect the link between Greek-key motifs 2 and 3, whereas both Crygbnop and Cryget mutations are supposed to truncate the fourth Greek-key motif. All three mutations are predicted to alter protein folding of the gamma-crystallins and result in lens cataract, but the phenotype for each is quite distinctive.

Amino Acid Sequence↗
PubMed1993

Mutational analysis of the fission yeast p34cdc2 protein kinase gene.

The p34cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34cdc2 (each ca. 60% identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with tryptophan renders the resulting mutant protein p80cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34cdc2 function. Five acidic amino acids have also been mutated within p34cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34cdc2 enzyme.

Amino Acid Sequence↗
PubMed1997

Identification and genetic characterization of the Pseudomonas aeruginosa leuB gene encoding 3-isopropylmalate dehydrogenase.

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-beta-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway.

3-Isopropylmalate Dehydrogenase↗
PubMed2000

The spectrum of mutations in erythrokeratodermias--novel and de novo mutations in GJB3.

Intercellular channels in skin are a complex and functionally diverse system formed by at least eight connexins (Cx). Our recent molecular studies implicating Cx defects in inherited skin disorders emphasize the critical role of this signaling pathway in epidermal differentiation. Erythrokeratodermia variabilis (EKV) is an autosomal dominant genodermatosis with a striking phenotype characterized by the independent occurrence of transient localized erythema and hyperkeratosis. The disease maps to 1p34-p35, and recently we identified the causative gene GJB3 encoding Cx31. We have now investigated GJB3 in two families and three sporadic cases with EKV, and report three new heterozygous mutations. In a sporadic case, we detected a mutation leading to substitution of a conserved phenylalanine (F137L) in the third transmembrane domain, which likely interferes with the proper assembly or gating properties of connexons. In another family, all three affected individuals carried two distinct mutations on the same GJB3 allele. However, only a de novo heterozygous missense mutation replacing arginine 42 with proline (R42P) co-segregated with the disease, while a 12 bp deletion predicted to eliminate four amino acid residues in the variable carboxy terminal domain of Cx31 was also found in clinically unaffected relatives but not in 90 unaffected controls. Including the previously published mutations, in toto, five different missense mutations have now been detected in 6 out of 17 families investigated by our laboratory, all of which presumably affect the cytoplasmic amino terminal and transmembrane domains of Cx31. In contrast, two mutations linked to progressive high-tone hearing impairment were located in the second extracellular domain, suggesting that the character and position of Cx mutations determine their phenotypic expression in different tissues. However, the phenotypic spectrum of GJB3 mutations seems not to include progressive symmetric erythrokeratodermia, another dominant genodermatosis with overlapping features, since no mutations were found in six unrelated families tested.

Connexins↗
PubMed1999

Characterization of a PDR1 mutant allele from a clotrimazole-resistant sake yeast mutant with improved fermentative activity.

Clotrimazole-resistant mutants from various sake yeasts show improved fermentative activity in sake mash while retaining their parental advantages for sake making. These mutants also exhibit pleiotropic drug resistance (PDR) phenotypes. To investigate the relationship between the improvement of fermentative activity and PDR phenotypes, a PDR1 mutant allele (pdr1-h176) encoding a transcription factor was cloned from a clotrimazole-resistant mutant, HL176 (MATa/MATalpha), using PCR amplification. The nucleotide sequences of pdr1-h176 and its wild allele were determined. The mutant allele contained a missense point mutation (L309S) that can confer a PDR phenotype on yeast. This amino acid substitution is located in the conserved motif II in the inhibitory domain of Pdr1p, and is very close to the cluster of three mutation points (P298A, K302Q, and M308I) described by Carvajal et al. (Mol. Gen. Genet., 256, 406-415, 1997) in laboratory strains. A PDR1 wild allele of HL163, the parent strain of HL176, was replaced by pdr1-h176 using gene recombination at the homologous site. The resultant transformants (PDR1/pdr1-h176) showed the same PDR phenotype as HL176, and they fermented sake mash efficiently even in the final fermentation stage, while HL163 did not. The amino acid substitution (L309S) in pdr1-h176 was considered to be sufficient to improve the fermentative activity of sake yeast, in addition to conferring the PDR phenotype.

Journal Article↗
PubMed2005

Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA.

Congenital central hypoventilation syndrome, also known as Ondine's curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.

Adult↗
PubMed1995

Double replacement gene targeting for the production of a series of mouse strains with different prion protein gene alterations.

We have developed a double replacement gene targeting strategy which enables the production of a series of mouse strains bearing different subtle alterations to endogenous genes. This is a two-step process in which a region of the gene of interest is first replaced with a selectable marker to produce an inactivated allele, which is then re-targeted with a second vector to reconstruct the inactivated allele, concomitantly introducing an engineered mutation. Five independent embryonic stem cell lines have been produced bearing different targeted alterations to the prion protein gene, including one which raises the level of expression. We have constructed mice bearing the codon 101 proline to leucine substitution linked to the human familial prion disease, Gerstmann-Straussler-Scheinker syndrome. We anticipate that this procedure will have applications to the study of human inherited diseases and the development of therapies.

Alleles↗