Photorepair of ultraviolet-induced petite mutational damage in Saccharomyces cerevisiae requires the product of the PHR1 gene.

A wild-type (phr+) diploid yeast strain showed photorepair of petite mutational damage, whereas a photoreactivation-deficient (phr1/phr1) diploid strain did not, indicating that the PHR1 gene product was required for mitochondrial photorepair.     

qUVR-10, a major quantitative trait locus for ultraviolet-B resistance in rice, encodes cyclobutane pyrimidine dimer photolyase

Rice qUVR-10, a quantitative trait locus (QTL) for ultraviolet-B (UVB) resistance on chromosome 10, was cloned by map-based strategy. It was detected in backcross inbred lines (BILs) derived from a cross between the japonica variety Nipponbare (UV resistant) and the indica variety Kasalath (UV sensitive). Plants homozygous for the Nipponbare allele at the qUVR-10 locus were more resistant to UVB compared with the Kasalath allele. High-resolution mapping using 1850 F(2) plants enabled us to delimit qUVR-10 to a <27-kb genomic region. We identified a gene encoding the cyclobutane pyrimidine dimer (CPD) photolyase in this region. Activity of CPD photorepair in Nipponbare was higher than that of Kasalath and nearly isogenic with qUVR-10 [NIL(qUVR-10)], suggesting that the CPD photolyase of Kasalath was defective. We introduced a genomic fragment containing the CPD photolyase gene of Nipponbare to NIL(qUVR-10). Transgenic plants showed the same level of resistance as Nipponbare did, indicating that the qUVR-10 encoded the CPD photolyase. Comparison of the qUVR-10 sequence in the Nipponbare and Kasalath alleles revealed one probable candidate for the functional nucleotide polymorphism. It was indicated that single-base substitution in the CPD photolyase gene caused the alteration of activity of CPD photorepair and UVB resistance. Furthermore, we were able to develop a UV-hyperresistant plant by overexpression of the photolyase gene.     

Cloning, sequencing, expression and characterization of DNA photolyase from Salmonella typhimurium.

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.     

Trichoderma atroviride PHR1, a fungal photolyase responsible for DNA repair, autoregulates its own photoinduction

The photolyases, DNA repair enzymes that use visible and long-wavelength UV light to repair cyclobutane pyrimidine dimers (CPDs) created by short-wavelength UV, belong to the larger photolyase-cryptochrome gene family. Cryptochromes (UVA-blue light photoreceptors) lack repair activity, and sensory and regulatory roles have been defined for them in plants and animals. Evolutionary considerations indicate that cryptochromes diverged from CPD photolyases before the emergence of eukaryotes. In prokaryotes and lower eukaryotes, some photolyases might have photosensory functions. phr1 codes for a class I CPD photolyase in Trichoderma atroviride. phr1 is rapidly induced by blue and UVA light, and its photoinduction requires functional blue light regulator (BLR) proteins, which are White Collar homologs in Trichoderma. Here we show that deletion of phr1 abolished photoreactivation of UVC (200 to 280 nm)-inhibited spores and thus that PHR1 is the main component of the photorepair system. The 2-kb 5' upstream region of phr1, with putative light-regulated elements, confers blue light regulation on a reporter gene. To assess phr1 photosensory function, fluence response curves of this light-regulated promoter were tested in null mutant (Deltaphr1) strains. Photoinduction of the phr1 promoter in Deltaphr1 strains was >5-fold more sensitive to light than that in the wild type, whereas in PHR1-overexpressing lines the sensitivity to light increased about 2-fold. Our data suggest that PHR1 may regulate its expression in a light-dependent manner, perhaps through negative modulation of the BLR proteins. This is the first evidence for a regulatory role of photolyase, a role usually attributed to cryptochromes.     

Marker-assisted selection of restored male-fertile Brassica napus plants using a set of dominant RAPD markers

Bulked segregant analysis was used to identify RAPD markers in oilseed rape (Brassica napus L.) that were linked to a male fertility restorer gene for Ogura cytoplasmic male sterility. After screening for polymorphisms using 960 primers, 14 RAPD markers were mapped to a 25 cM region including the restorer locus, a mapping population of 242 F₂ individuals being employed. The map was used to select 11 markers that were investigated for polymorphisms between the restorer donor line and 46 recipient lines. A set of four RAPD markers, one in coupling phase with the restorer allele and three with the non-restorer allele, which were informative in all 46 combinations, were used in marker assisted selection of plants homozygous for the restorer allele. A total of 906 homozygous restored plants were found among the 4605 BC₁F₂ plants analysed. Phenotypic data of a subset of the classified plants was compared with the RAPD data and the expected number of recombinants was calculated from the map data. A close correspondence between the expected and observed numbers of plants with a deviating phenotype was found. Thus, use of a set of dominant RAPD markers provides a way obtaining reliable data for marker-assisted selection.     

Cloning and mapping of Saccharomyces cerevisiae photoreactivation gene PHR1.

The yeast Saccharomyces cerevisiae, like most organisms, is able to directly repair pyrimidine dimers by using a photoreactivating enzyme and visible light. Cells carrying the phr1 mutation were shown previously to be unable to photoreactivate dimers, but neither the map position nor the primary gene product of the PHR1 gene has been determined. We have cloned this gene and determined its map position. A plasmid containing a 6.4-kilobase yeast DNA insert has been isolated and shown to restore photoreactivation in a phr1 strain. A 3.1-kilobase subclone has also been shown to complement phr1. The original plasmid was targeted to integrate into chromosomal DNA at a site homologous to the insert by cutting within the insert. Two of these integrants have been mapped on the right arm of chromosome XV; the integrants have been further mapped at ca. 13 centimorgans from prt1. It has also been independently determined that phr1 maps at this location. Thus, we have determined the map position of PHR1 and also have shown that the plasmid contains PHR1 rather than a suppressor of the phr1 mutation.     

Purification and characterization of three members of the photolyase/cryptochrome family blue-light photoreceptors from Vibrio cholerae

The sequence of Vibrio cholerae genome revealed three genes belonging to the photolyase/cryptochrome blue-light photoreceptor family. The proteins encoded by the three genes were purified and characterized. All three proteins contain folate and flavin cofactors and have absorption peaks in the range of 350-500 nm. Only one of the three, VcPhr, is a photolyase specific for cyclobutane pyrimidine dimers. The other two are cryptochromes and were designated VcCry1 and VcCry2, respectively. Mutation of phr abolishes photoreactivation of UV-induced killing, whereas mutations in cry1 and cry2 do not affect photorepair activity. VcCry1 exhibits some unique features. Of all cryptochromes characterized to date, it is the only one that contains stoichiometric amounts of both chromophores and retains its flavin cofactor in the two-electron reduced FADH2 form. In addition, VcCry1 exhibits RNA binding activity and co-purifies with an RNA of 60-70 nucleotides in length.     

QTL analysis for pseudostem pungency in bunching onion (Allium fistulosum)

Low pungency is one of the most important agronomic traits in bunching onion (Allium fistulosum L.). Although the degree of pungency can be evaluated indirectly using a colorimetric test for pyruvic acid, DNA markers linked to low-pungency quantitative trait loci (QTLs) are still desired. In this study, we evaluated pungency in the bunching onion pseudostem through six trials conducted over 3?years using an F _2:3 population. QTL analysis based on the genetic linkage map revealed that the major pungency QTL was located within a 24.2-cM interval on Chr. 2a. The low-pungency parent-derived allele at AFAT04B03, a simple sequence repeat locus linked to the pungency QTL, was rare among commercial bunching onion cultivars. In addition, individuals homozygous for the low-pungency parent-derived allele at AFAT04B03 were significantly less pungent than those that were homozygous or heterozygous. Thus, these findings suggest that AFAT04B03 is an effective selection marker for low pungency in bunching onion breeding.     

The Abnormal Oocyte Phenotype Is Correlated with the Presence of Blood Transposon in Drosophila Melanogaster

The abnormal oocyte mutation (2;44) originates in the wild: it confers no visible phenotype on homozygous abo males or females, but homozygous abo females produce defective eggs and the probability of their developing into adults is much lower than that of heterozygous sister females. We isolated by chromosome walking 200 kb of DNA from region 32. This paper reports that a restriction enzyme site polymorphism analysis in wild type and mutant stocks allowed us to identify a DNA rearrangement present only in stocks carrying the abo mutation. The rearrangement is caused by a DNA insert on the abo chromosome in region 32E which, by restriction map and sequence analysis, was identified as copia-like blood transposon. The transposon, in strains that had remained in abo homozygous conditions for several generations and had lost the abo maternal-effect, was no longer present in region 32E. Certain features of the abo mutation, discussed in the light of this finding, may be ascribed to the nature of the particular allele studied.     

酵母糖化酶基因高效表达的剂量效应研究

通过原生质体融合和秋水仙素加倍技术,将糖化酵母的３个等效异位基因分别构建成交配型位点等痊基因纯合和糖化酶基因各自纯各加倍的纯合二倍体（ＳＴＡ１／ＳＴＡ１、ＳＴＡ２／ＳＴＡ２和ＳＴＡ３ＳＴＡ３）以及糖化酶基因相互组织加倍的组合二倍体（ＳＴＡ１／ＳＴＡ２、ＳＴＡ２／ＳＴＡ３和ＳＴＡ１／ＳＴＡ３）。研究了这３个糖化酶基因表达的剂量效及其相互关系。糖化酶酶活测定的结果表明,在交配型位点等位基因纯合状态下,     

Curly bare (cub), a new mouse mutation on chromosome 11 causing skin and hair abnormalities,and a modifier gene (mcub) on chromosome 5

In the outcrossing of a new recessive mouse mutation causing hair loss, a new wavy-coated phenotype appeared. The two distinct phenotypes were shown to be alternative manifestations of the same gene mutation and attributable to a single modifier locus. The new mutation, curly bare (cub), was mapped to distal Chr 11 and the modifier (mcub) was mapped to Chr 5. When homozygous for the recessive mcub allele, cub/cub mice appear hairless. A single copy of the dominant Mcub allele confers a full, curly coat in cub/cub mice. Reciprocal transfer of full-thickness skin grafts between mutant and control animals showed that the skin phenotype was tissue autonomous. The hairless cub/cub mcub/mcub mice show normal contact sensitivity responses to oxazolone. The similarity of the wavy coat phenotype to those of Tgfa and Egfr mutations and the map positions of cub and mcub suggest candidate genes that interact in the EGF receptor signal transduction pathway.     

Minisatellite "isoallele" discrimination in pseudohomozygotes by single molecule PCR and variant repeat mapping.

The D1S8 hypervariable minisatellite MS32 has a heterozygosity of 97.5% based on detectable differences in allele length using standard Southern blot analysis. It has previously been shown that the basic repeat unit is in itself variable and that this may be used to map the internal structure of an allele. This method has already been used to establish that alleles of the same length may have differing internal structures between nonrelated individuals. We now extend this approach to demonstrate that two apparently homozygous individuals are in fact heterozygotes. For each individual the two comigratory alleles were separated, without cloning, using single molecule dilution (SMD) of genomic DNA and recovery with PCR. Mapping of the variant repeat units revealed highly diverged internal structures and, for one individual, a size difference of one repeat unit (29 bp). SMD and PCR recovery provide an efficient system for separating comigratory alleles without prerequirement for knowledge of sequence differences.     

Differential interleukin-10 production stratified by -571 promoter polymorphism in purified human immune cells

A C-to-A base substitution has been identified at bp −571 in the IL-10 promoter and has been linked to numerous diseases. To investigate the role of this polymorphism on IL-10 production, T cells, B cells and monocytes were enriched from peripheral blood from subjects either homozygous for the C or A allele. Treatment of monocytes and B cells with lipopolysaccharide from individuals homozygous for the C allele resulted in higher levels of IL-10 production as compared to monocytes from individuals homozygous for the A allele. Though not statistically significant, when B cells were treated with anti-IgM or T cells with concanavalin A higher levels of IL-10 were produced from individuals homozygous for the A allele. Changes in IL-10 protein production were paralleled by similar changes in IL-10 mRNA production. These results demonstrate that changes in IL-10 production observed due to the −571 genotype depend on both cell type and stimulus.     

Segregation of microsatellite alleles and residual heterozygosity at single loci in homozygous androgenetic common carp (Cyprinus carpio L.).

Thirty-three androgenetic progeny groups of common carp were analysed using 11 microsatellite markers to (i) verify the homozygous status of the 566 androgenetic individuals, (ii) analyse the microsatellite allele segregation, and (iii) study the possible association of microsatellite alleles with phenotypic traits. In total, 92% of the androgenetic individuals proved to be homozygous at all 11 loci. Forty-three of the 47 heterozygous individuals were heterozygous at a single locus only. This heterozygosity was probably due to DNA fragments caused by UV irradiation of the eggs. although the maternal origin of the fragments could not be proved beyond doubt. Screening with 11 microsatellites also revealed two linkage groups, a segregation distortion at two microsatellite loci, and the possible association of some microsatellites with mass, length, stress-related plasma cortisol levels, and basal plasma glucose levels. The success of the linkage and association study could be explained by a low recombination frequency due to high chiasma interference. This would imply a relatively short genetic map for common carp.     

Trade-offs between male and female reproduction associated with allozyme variation in phosphoglucoisomerase in an annual plant (Clarkia unguiculata: Onagraceae)

The genotype of an individual for allozymes such as phosphoglucoisomerase (Pgi) is often not neutral with regard to fitness. Studies of several taxa have found consistent fitness differences among Pgi genotypes expressing different allozymes. We conducted a greenhouse experiment with Clarkia unguiculata to determine whether allelic variation at the Pgi-C1 locus may affect components of male and female function. We found significant differences in siring success between pollen donors homozygous for different Pgi alleles. When a mixture of pollen was applied to stigmas under conditions of gametophytic competition (more pollen deposited on stigmas than there are ovules available to fertilize), donors homozygous for the C allele of Pgi sired more seeds per fruit than B-allele donors. Differences between genotypes with respect to female fertility per fruit contrasted with the male advantage associated with the C allele. Recipients homozygous for the C allele produced fruits with more aborted seeds and fewer viable seeds than recipients homozygous for the B allele. These results suggest that allelic variation at a single locus may have opposing effects on male and female reproductive success in C. unguiculata, and that trade-offs between the two types of reproductive success could contribute to the maintenance of variation at the Pgi-C1 locus.     

Electrophoretic transferrin variation in fur seals (Arctocephalus spp.) at Marion Island

1. Variation in transferrin types were investigated in sympatric populations of two fur seal species which are undergoing limited hybridization at Marion Island. 2. Vertical polyacrylamide gel electrophoresis of serum was performed to demonstrate the transferrin types. 3. The two species appear fixed for alternative alleles: A. tropicalis being homozygous for the fast allele, and A. gazella (with one exception) being homozygous for the slow allele, indicating low gene flow between these two species. The single hybrid tested was homozygous for the slow allele. 4. The use of electrophoretically determined transferrin variation holds promise for the investigation of these and other sympatric fur seal populations.     

Mutations in the arylsulfatase A pseudodeficiency allele causing metachromatic leukodystrophy.

We identified a patient suffering from late infantile metachromatic leukodystrophy who genetically seemed to be homozygous for the mutations signifying the arylsulfatase A pseudodeficiency allele. Homozygosity for the pseudodeficiency allele is associated with low arylsulfatase A activity but does not cause a disease. Analysis of the arylsulfatase A gene in this patient revealed a C----T transition in exon 2, causing a Ser 96----Phe substitution in addition to the sequence alterations causing arylsulfatase A pseudodeficiency. Although this mutation was found only in 1 of 78 metachromatic leukodystrophy patients tested, five more patients were identified who seemed hetero- or homozygous for the pseudodeficiency allele. The existence of nonfunctional arylsulfatase A alleles derived from the pseudodeficiency allele calls for caution when the diagnosis of arylsulfatase A pseudodeficiency is based solely on the identification of the mutations characterizing the pseudodeficiency allele.     

NFKB1 promoter variation implicates shear-induced NOS3 gene expression and endothelial function in prehypertensives and stage I hypertensives

In endothelial cells, NF-kappaB is an important intracellular signaling molecule by which changes in wall shear stress are transduced into the nucleus to initiate downstream endothelial nitric oxide synthase (NOS3) gene expression. We investigated whether NF-kappa light-chain gene enhancer in B cells 1 (NFKB1) promoter polymorphism ((-94)NFKB1 I/D, where I is the insertion allele and D is the deletion allele) was associated with 1) NOS3 gene expression in endothelial cells under physiological levels of unidirectional laminar shear stress (LSS) and 2) endothelial function in prehypertensive and stage I hypertensive individuals before and after a 6-mo supervised endurance exercise intervention. Competitive EMSAs revealed that proteins present in the nuclei of endothelial cells preferentially bound to the I allele NFKB1 promoter compared with the D allele. Reporter gene assays showed that the I allele promoter had significantly higher activity than the D allele. In agreement with these observations, homozygous II genotype cells had higher p50 expression levels than homozygous DD genotype cells. Cells with the homozygous II genotype showed a greater increase in NOS3 protein expression than did homozygous DD genotype cells under LSS. Functional experiments on volunteers confirmed higher baseline reactive hyperemic forearm blood flow, and, furthermore, the subgroup analysis revealed that DD homozygotes were significantly less prevalent in the exercise responder group compared with II and ID genotypes. We conclude that the (-94)NFKB1 I/D promoter variation contributes to the modulation of vascular function and adaptability to exercise-induced flow shear stress, most likely due to differences in NFKB1 gene transactivity.