Induction and decline of HPRT mutants and deletions following a low dose radiation exposure at Chernobyl

This study was conducted to evaluate the ability of mutation in the hypoxanthine-phosphoribosyltransferase gene (HPRT) to detect radiation-induced mutation in lymphocytes of Russian Chernobyl Clean-up workers, particularly as a function of time after exposure. It is part of a multi-endpoint study comparing HPRT mutation with chromosome translocation and glycophorin A mutation [Radiat. Res. 148 (1997) 463], and extends an earlier report on HPRT [Mutat. Res. 431 (1999) 233] by including data from all 9 years of our study (versus the first 6 years) and analysis of deletion size. Blood samples were collected from 1991 to 1999. HPRT mutant frequency (MF) as determined by the cloning assay was elevated 16% in Clean-up workers (N=300, the entire group minus one outlier) compared to Russian Controls (N=124) when adjusted for age and smoking status (P=0.028). Since exposures occurred over a short relative to the long sampling period, the year of sampling corresponded roughly to the length of time since exposure (correlation coefficient=0.94). When date of blood sample was considered, Control MF was not time dependent. Clean-up worker MF was estimated to be 47% higher than Control MF in 1991 (P=0.004) and to decline 4.4% per year thereafter (P=0.03). A total of 1123 Control mutants and 2799 Clean-up worker mutants were analyzed for deletion type and size by PCR assay for retention of HPRT exons and flanking markers on the X chromosome. There was little difference between the overall deletion spectra of Clean-up workers and Controls. However, there was a decline in the average size of deletions of Clean-up workers as time after exposure at Chernobyl increased from 6 to 13 years (P< or =0.05). The results illustrate the sensitivity of HPRT somatic mutation as a biomarker for populations with low dose radiation exposure, and the dependence of this sensitivity on time elapsed since radiation exposure.     

Characteristics of the end-joining of DNA double-strand breaks by the ataxia-telangiectasia nuclear extract

A double-strand break was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to Escherichia coli cells and treated with nuclear extracts from human cells. The efficiency of rejoining was monitored by Southern blot analysis and the fidelity of rejoining was measured by expressing the ccdB gene after bacterial transformation. The efficiency of rejoining in the nuclear extract from an ataxia-telangiectasia (A-T) cell line was comparable to that from a control cell line. However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract. All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions. The deletion spectrum caused by the A-T nuclear extract was distinct from that of the control extract. These results indicate that the ccdB gene is useful for analysis of mis-rejoining and that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA.     

Phenotypic assessment and mapped markers for<Emphasis Type="Italic"> H31,</Emphasis> a new wheat gene conferring resistance to Hessian fly (Diptera: Cecidomyiidae)

A new source of resistance to the highly virulent and widespread biotype L of the Hessian fly, Mayetiola destructor (Say), was identified in an accession of tetraploid durum wheat, Triticum turgidum Desf., and was introgressed into hexaploid common wheat, Triticum aestivum L. Genetic analysis and deletion mapping revealed that the common wheat line contained a single locus for resistance, H31, residing at the terminus of chromosome 5BS. H31 is the first Hessian fly-resistance gene to be placed on 5BS, making it unique from all previously reported sources of resistance. AFLP analysis identified two markers linked to the resistance locus. These markers were converted to highly specific sequence-tagged site markers. The markers are being applied to the development of cultivars carrying multiple genes for resistance to Hessian fly biotype L in order to test gene pyramiding as a strategy for extending the durability of deployed resistance.Communicated by J. Dvorak     

Comparative mapping of the wheat chromosome 5A Vrn-A1 region with rice and its relationship to QTL for flowering time

 The vernalization gene Vrn-A1 on chromosome 5A is the predominant gene determining the spring/winter habit difference in bread wheat. Vrn-A1 was physically mapped using a set of deletion lines which located it to the region of chromosome 5A flanked by deletion breakpoints 0.68 and 0.78. This interval was shown to be homoeologous to a region of rice chromosome 3 that contains the flowering-time QTL Hd-6, previously mapped in a Nipponbare×Kasalath cross, and FLTQ1, a novel QTL identified by analysis of 78 F₃ families derived from a cross of ‘IR20’ב63–83’. Possible relationships between Vrn-A1 and rice QTL are discussed. Analysis of the chromosome 5A deletion lines showed evidence for a second, more proximal flowering-time effect located between deletion breakpoints 0.56 and 0.64. The proximal part of chromosome 5A is homoeologous to rice chromosome 9, on which two QTL were detected in the ‘IR20ב63–83’ cross. The possible relationship between these effects is also discussed. Received: 23 December 1997 / Accepted: 12 January 1998     

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Δ52 in an αC–βC loop. Double deletion mutants showed that the loop αC–βC has nonadditive couplings with the βF–βG and βG–βH loops regarding stability. Single deletion to the loops αC–βC or βC–βD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.     

Indel evolution of mammalian introns and the utility of non-coding nuclear markers in eutherian phylogenetics

Nuclear DNA intron sequences are increasingly used to investigate evolutionary relationships among closely related organisms. The phylogenetic usefulness of intron sequences at higher taxonomic levels has, however, not been firmly established and very few studies have used these markers to address evolutionary questions above the family level. In addition, the mechanisms driving intron evolution are not well understood. We compared DNA sequence data derived from three presumably independently segregating introns (THY, PRKC I and MGF) across 158 mammalian species. All currently recognized extant eutherian mammalian orders were included with the exception of Cingulata, Dermoptera and Scandentia. The total aligned length of the data was 6366 base pairs (bp); after the exclusion of autapomorphic insertions, 1511 bp were analyzed. In many instances the Bayesian and parsimony analyses were complementary and gave significant posterior probability and bootstrap support (>80) for the monophyly of Afrotheria, Euarchontoglires, Laurasiatheria and Boreoeutheria. Apart from finding congruent support when using these methods, the intron data also provided several indels longer than 3 bp that support, among others, the monophyly of Afrotheria, Paenungulata, Ferae and Boreoeutheria. A quantitative analysis of insertions and deletions suggested that there was a 75% bias towards deletions. The average insertion size in the mammalian data set was 16.49 bp +/- 57.70 while the average deletion was much smaller (4.47 bp +/- 14.17). The tendency towards large insertions and small deletions is highlighted by the observation that out of a total of 17 indels larger than 100 bp, 15 were insertions. The majority of indels (>60% of all events) were 1 or 2 bp changes. Although the average overall indel substitution rate of 0.00559 per site is comparable to that previously reported for rodents and primates, individual analyses among different evolutionary lineages provide evidence for differences in the formation rate of indels among the different mammalian groups.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.     

高效删除标记基因的Bhlea2植物表达载体的构建

为培育去除选择标记基因的耐旱转基因植物,同时利用Cre/Lox和FLP/frt系统,构建一个能够高效删除标记基因的Bhlea2基因植物表达载体.拟南芥rd29A启动子是在低温、干旱、高盐胁迫下的快速应答启动子,玉米ubiquitin启动子可有效驱动外源基因的转录,拟南芥pAB5启动子是花粉及胚胎等发育早期特异表达的启动子,利用上述启动子构建了表达Bhlea2基因并能够删除标记基因的植物表达载体.该表达载体包括重组酶表达元件pAB5-FLP、Bhlea2抗旱基因表达元件rd29A-Bhlea2和bar标记基因表达元件ubiquitin-bar.     

Why Are Young and Old Repetitive Elements Distributed Differently in the Human Genome?

Alu elements are not distributed homogeneously throughout the human genome: old elements are preferentially found in the GC-rich parts of the genome, while young Alus are more often found in the GC-poor parts of the genome. The process giving rise to this differential distribution remains poorly understood. Here we investigate whether this pattern could be due to a preferential degradation of Alu elements integrated in GC-poor regions by small indel mutations. We aligned 5.1 Mb of human and chimpanzee sequences and examined whether the rate of insertion and deletion inside Alu elements differed according to the base composition surrounding them. We found that Alu elements are not preferentially degraded in GC-poor regions by indel events. We also looked at whether very young L1 elements show the same change in distribution compared to older ones. This analysis indicated that L1 elements also show a shift in their distribution, although we could not assess it as precisely as for Alu elements. We propose that the differential distribution of Alu elements is likely to be due to a change in their pattern of insertion or their probability of fixation through evolutionary time.Reviewing Editor: Dr. Stephen Freeland