Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2

Elevated expression of the plasma membrane drug efflux pump proteins Cdr1p and Cdr2p was shown to accompany decreased azole susceptibility in Candida albicans clinical isolates. DNA sequence analysis revealed extensive allelic heterozygosity, particularly of CDR2. Cdr2p alleles showed different abilities to transport azoles when individually expressed in Saccharomyces cerevisiae. Loss of heterozygosity, however, did not accompany decreased azole sensitivity in isogenic clinical isolates. Two adjacent non-synonymous single nucleotide polymorphisms (NS-SNPs), G1473A and I1474V in the putative transmembrane (TM) helix 12 of CDR2, were found to be present in six strains including two isogenic pairs. Site-directed mutagenesis showed that the TM-12 NS-SNPs, and principally the G1473A NS-SNP, contributed to functional differences between the proteins encoded by the two Cdr2p alleles in a single strain. Allele-specific PCR revealed that both alleles were equally frequent among 69 clinical isolates and that the majority of isolates (81%) were heterozygous at the G1473A/I1474V locus, a significant (P < 0.001) deviation from the Hardy-Weinberg equilibrium. Phylogenetic analysis by maximum likelihood (Paml) identified 33 codons in CDR2 in which amino acid allelic changes showed a high probability of being selectively advantageous. In contrast, all codons in CDR1 were under purifying selection. Collectively, these results indicate that possession of two functionally different CDR2 alleles in individual strains may confer a selective advantage, but that this is not necessarily due to azole resistance.     

A mutation that permits the expression of normally silent copies of mating-type information in Saccharomyces cerevisiae

Studies of heterothallic and homothallic strains of Saccharomyces cerevisiae have led to the suggestion that mating-type information is located at three distinct sites on chromosome 3, although only information at the mating-type (MAT) locus is expressed (Hicks, Strathern and Herskowitz, 1977). We have found that the recessive mutation cmt permits expression of the normally silent copies of mating-type information at the HMa and HM alpha loci. In haploid strains carrying HMa and HM alpha, the cmt mutation allows the simultaneous expression of both a and alpha information, leading to a nonmating ("MATa/MAT alpha") phenotype. The effects of cmt can be masked by changing the mating-type information at HMa or HM alpha. For example, a cell of genotype MATa hma HM alpha cmt has an a mating type, while a MAT alpha hma HM alpha cmt strain is nonmating. Expression of mating-type information at the HM loci can correct the mating and sporulation defects of the mata* and mat alpha 10 alleles. Meiotic segregants recovered from cmt/cmt diploids carrying the mat mutations demonstrate that these mutants are not "healed" to normal MAT alleles, as is the case in parallel studies using the homothallism gene HO.--All of the results are consistent with the notion that the HMa and hm alpha alleles both code for alpha information, while HM alpha and hma both code for a information. The cmt mutation demonstrates that these normally silent copies of mating-type and sporulation information can be expressed and that the information at these loci is functionally equivalent to that found at MAT. The cmt mutation does not cause interconversions of mating-type alleles at MAT, and it is not genetically linked to MAT, HMa, HM alpha or HO. In cmt heterozygotes, cmt becomes homozygous at a frequency greater than 1% when the genotype at the MAT locus is mata*/MAT alpha or mat alpha 10/MATa.     

Dis1: A Yeast Gene Required for Proper Meiotic Chromosome Disjunction

Mutants at a newly identified locus, DIS1 (disjunction), were detected by screening for mutants that generate aneuploid spores (chromosome VIII disomes) at an increased frequency. Strains carrying the partially dominant alleles, DIS1-1 or DIS1-2, generate disomes at rates up to 100 times the background level. Mitotic nondisjunction is also increased 10- to 50-fold over background. Half-tetrad analysis of disomes for a marked interval on chromosome VIII yields wild-type map distances, indicating that a general recombination deficiency is not the cause of nondisjunction. Meiotic nondisjunction in DIS1 mutants is not chromosome specific; 5% of the spores disomic for chromosome VIII are also disomic for chromosome III. Although only one disomic spore is found per exceptional ascus most of the disomes appear to be generated in the first meiotic division because recovered chromosome VIII disomes contain mostly nonsister chromosomes. We propose that disome generation in the DIS1 mutants results from precocious separation of sister centromeres.     

Developmental changes in the human heavy chain CDR3

The CDR3 of the Ig H chain (CDR3(H)) is significantly different in fetal and adult repertoires. To understand the mechanisms involved in the developmental changes in the CDR3(H) of Ig H chains, sets of nonproductive V(H)DJ(H) rearrangements obtained from fetal, full-term neonates and adult single B cells were analyzed and compared with the corresponding productive repertoires. Analysis of the nonproductive repertoires was particularly informative in assessing developmental changes in the molecular mechanisms of V(H)DJ(H) recombination because these rearrangements did not encode a protein and therefore their distribution was not affected by selection. Although a number of differences were noted, the major reasons that fetal B cells expressed Ig H chains with shorter CDR3(H) were both diminished TdT activity in the DJ(H) junction and the preferential use of the short J(H) proximal D segment D7-27. The enhanced usage of D7-27 by fetal B cells appeared to relate to its position in the locus rather than its short length. The CDR3(H) progressively acquired a more adult phenotype during ontogeny. In fetal B cells, there was decreased recurrent DJ(H) rearrangements before V(H)-DJ(H) rearrangement and increased usage of junctional microhomologies both of which also converted to the adult pattern during ontogeny. Overall, these results indicate that the decreased length and complexity of the CDR3(H) of fetal B cells primarily reflect limited enzymatic modifications of the joins as well as a tendency to use proximal D and J(H) segments during DJ(H) rearrangements.     

High-affinity, peptide-specific T cell receptors can be generated by mutations in CDR1, CDR2 or CDR3

The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.     

Genetic polymorphism of human factor H (beta 1H)

Human Factor H (beta 1H) was found to be polymorphic after neuraminidase treatment and isoelectric focusing (IEF) under completely denaturing conditions. Three variants, FH 1, FH 2, and FH 3, were identified in a sample population of 81 unrelated caucasoid individuals. Family studies demonstrated correct mendelian segregation of FH 1, FH 2, and FH 3. Our data indicate that these genetic variants of human Factor H are encoded by three codominant alleles, FH*1, FH*2, and FH*3, at a single autosomal locus FH. In the sample analyzed, the gene frequencies of FH*1, FH*2, and FH*3 were, respectively, 0.691, 0.302, and 0.006.     

A Suppressor of a Mating-Type Limited Zygotic Lethal Allele Also Suppresses Uniparental Chloroplast Gene Transmission in Chlamydomonas Monoica

Uniparental inheritance of Chlamydomonas chloroplast genes is thought to involve modification of maternal (mt(+)) chloroplast genomes to protect against a nuclease that is activated after gamete fusion. The mating-type limited mtl-1 mutant strain of Chlamydomonas monoica is unable to protect mt(+)-derived chloroplast DNA. Zygotes homozygous for mtl-1 lose all chloroplast DNA and fail to germinate. We have selected for suppression of this zygote-specific lethality, and have obtained 20 mutant strains that produce viable homozygotes despite the continued presence of the mtl-1 allele. Genetic analysis indicates that the suppressor mutations are all recessive alleles at a single locus (sup-1) which is unlinked to mtl-1. Crosses between sup-1 strains carrying distinctive chloroplast antibiotic resistance markers also show predominantly biparental chloroplast gene transmission. Chloroplast nucleoids of both parental origins (stained with the DNA-specific fluorochrome, DAPI) are retained in the zygotes homozygous for sup-1. The data are compatible with the idea that the sup-1 (suppressor of uniparental inheritance) locus may encode a chloroplast DNA nuclease that is expressed from both parental genomes.     

A common drug-responsive element mediates the upregulation of the Candida albicans ABC transporters CDR1 and CDR2, two genes involved in antifungal drug resistance

Upregulation of the ATP-binding cassette (ABC) transporter genes CDR1 and CDR2 (Candida drug resistance 1 and 2) is a common mechanism observed in Candida albicans clinical isolates developing resistance to the class of azole antifungals. In this work, the regulatory elements of both genes were delimited using a reporter system in an azole-susceptible strain exposed to oestradiol, which allows transient induction of these genes. We found two regulatory elements in the CDR1 promoter: one responsible for basal expression (basal expression element; BEE) and the other required for oestradiol responsiveness (drug-responsive element I; DREI). In the CDR2 promoter, a single regulatory element responsible for oestradiol responsiveness (DREII) was detected. Both DREs shared a consensus of 21 bp with the sequence 5'-CGGA(A/T)ATCGGATATTTTTTTT-3' having no equivalent to known eukaryotic regulatory sequence. Consistent with this finding, two other C. albicans genes identified by a search for the presence of DRE in the C. albicans genome sequence database were responsive to oestradiol. Finally, the regulatory elements found in CDR1 and CDR2 were also functional in an azole-resistant strain with constitutive high expression of both transporters. These results suggest that, although CDR1 and CDR2 upregulation can be obtained by transient drug-induced and constitutive upregulation, these two processes converge to the same regulatory elements and probably mobilize the same trans-acting factors.     

Glutathione reductase activity deficiency in homozygous Gr1a1Neu mice does not cause haemolytic anaemia

A glutathione reductase (GR) mutant with approximately 50% residual enzyme activity in blood compared with wild-type was detected amongst offspring of isopropyl methanesulphonate-treated male mice. Homozygous mutants with only 2% residual enzyme activity were recovered in progeny of inter se matings of heterozygotes. Results of linkage studies indicate a mutation at the Gr1 structural locus on chromosome 8. The loss of GR activity was evident both in blood and in other tissue extracts. Erythrocyte and organo-somatic indices did not show differences between wild-types and homozygous mutants, indicating no association between the GR deficiency and haemolytic anaemia in this potential animal model.     

Mutations in the melanocortin 1 receptor (MC1R) gene are associated with coat colours in the domestic rabbit (Oryctolagus cuniculus)

We sequenced almost the complete coding region of the MC1R gene in several domestic rabbits (Oryctolagus cuniculus) and identified four alleles: two wild-type alleles differing by two synonymous single nucleotide polymorphisms (c.333A>G;c.555T>C), one allele with a 30-nucleotide in-frame deletion (c.304_333del30) and one allele with a 6-nucleotide in-frame deletion (c.280_285del6). A polymerase chain reaction-based protocol was used to distinguish the wild-type alleles from the other two alleles in 263 rabbits belonging to 37 breeds or strains. All red/fawn/yellow rabbits were homozygous for the c.304_333del30 allele. This allele represents the recessive e allele at the extension locus identified through pioneering genetic studies in this species. All Californian, Checkered, Giant White and New Zealand White rabbits were homozygous for allele c.280_285del6, which was also observed in the heterozygous condition in a few other breeds. Black coat colour is part of the standard colour in Californian and Checkered breeds, in contrast to the two albino breeds, Giant White and New Zealand White. Following the nomenclature established for the rabbit extension locus, the c.280_285del6 allele, which is dominant over c.304_333del30, may be allele E(D) or allele E(S).     

Genetic analysis of the non-sporulating phenotype of brewer's yeasts

The ploidies and sporulation abilities of six brewer's yeasts were examined. One (YB11-1) out of the six was triploid and sporulating, another (IFO2031) was haploid, and the others (IFO1167, IFO2003, S341 and YB3-7) were diploid and non-sporulating. The five non-sporulating strains did not have the premeiotic DNA synthesis. Their non-sporulating phenotypes were genetically analyzed by examining the sporulation abilities of hybrids between brewer's yeasts and standard genetic strains of Saccharomyces cerevisiae. All non-sporulating brewer's yeasts complemented 32 sporulation-deficient mutations (spoT–spoT23, spo1–spo5, spo7, spo8, spo10, and spo11). Hybrids between brewer's yeasts and haploid or diploid strains homozygous for the mating-type locus had poor or no sporualtion. On the contrary, hybrids between brewer's yeasts and diploid strains heterozygous for the mating-type locus sporulated at a high frequency. These results indicated that the non-sporulating phenotype of brewer's yeasts was caused by a deficiency of the mating-type genes rather than by mutations of sporulation genes. The Southern hybridization probed with the MATa gene showed polymorphisms in mating-type genes of brewer's yeasts.